Induction of ethoxy-resorufin-O-deethylase activity by halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons in primary hepatocytes of the green frog (Rana esculenta).

In this study, we measured the ethoxy-resorufin-O-deethylase (EROD) activity in primary hepatocytes of the common green frog Rana esculenta as a biomarker for cytochrome P4501A induction. We exposed hepatocytes derived from male and female frogs to several halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), polychlorinated biphenyls (PCB-126, PCB-118), and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), chrysene, anthracene, and pyrene. Exposure to PCB-118, anthracene, and pyrene, up to 1 microM, did not induce EROD activity, whereas TCDD and PCDF induced EROD activity maximally. In our primary frog hepatocytes, exposure to chrysene and BaP resulted in median effective concentration values (EC50) in the high nM range (82-1035 nM). Exposure to TCDD, PCDF, and PCB-126 resulted in EC50 values of 0.4 to 8, 0.07 to 0.7, and 3 to 133 nM, respectively, which is in the same range as EC50 values found in primary hepatocytes of birds. Compared to our frog hepatocytes, primary rat hepatocytes seem to be more sensitive to TCDD, chrysene, and BaP.

Reproductive failure and endocrine disruption by organohalogens in fish-eating birds.

Effects of organohalogens in fish-eating birds in the field have been monitored largely by studying reproductive outcome in contaminated populations or, at the individual level, by studying the sexual behaviour, egg production, or embryonal and postnatal development and survival. Endocrine disruption has been suggested as a mechanism, but proven cause-effect relationships between specific compounds and endocrine disruption with subsequent reduced reproductive outcome in fish-eating birds are scarce. The effects of organochlorines on hormone metabolism and circulating steroid levels are reviewed and discussed in relation to observed reproductive failures in the field.

Effects of furazolidone, PCB77, PCB126, Aroclor 1248, paraquat and p,p'-DDE on transketolase activity in embryonal chicken brain.

The effect of in ovo exposure to PCBs, DDE and paraquat on transketolase activity was measured in 19-day-old chicken embryos. Furazolidone was used as a positive control for decreased activity of the enzyme. The potency of contaminants to interact with transketolase was also tested in an in vitro system, using control brain 7000xg supernatants containing the enzyme. No effects were found on transketolase activity after in ovo or in vitro exposure to PCB126, Aroclor, DDE or paraquat. PCB77 decreased transketolase activity in vitro, but only at concentrations that, extrapolated to in ovo exposure, would be lethal to the embryo. Furazolidone decreased transketolase activity both in ovo and in vitro. For this contaminant, thiamine residues were analysed in the yolk sacs, but no differences were found between exposed and non-exposed eggs. Transketolase is dependent on thiamine pyrophosphate as a cofactor, and therefore, the decreased enzyme activity could be the result of an interaction between furazolidone and thiamine metabolism. Since thiamine residues were not affected by furazolidone and transketolase inhibition in vitro was similar to the inhibition after in ovo exposure, it was concluded that furazolidone interacted with transketolase on the enzymatic level rather than by a depletion of thiamine.

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).

In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in The Netherlands.

The ethoxy resorufin dealkylase (EROD) inducing potency of 10 polycyclic aromatic hydrocarbons (PAHs) is measured in the H4IIE in vitro bioassay and the results are compared to those reported in literature. The selected PAHs varied considerably in their potency to induce EROD activity. Anthracene (Ant) and phenanthrene (Phe) showed consistently no response. Naphthalene (Nap) showed no or a very weak response on EROD activity. Fluoranthene (Fla) and benzo[g,h,i]perylene (BghiP) showed weak responses at the highest doses. The other PAHs, including indeno[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), chrysene (Chr) and benzo[k]fluoranthene (BkF), showed full bell shaped dose-response curves. BaP EROD induction equivalency factors (BaP-1EF) were calculated and increased in the order Ant approximately Phe < Fla < Nap < BghiP < IP < BaA < BaP < Chr < BkF. Comparison of BaP-IEFs based on 50% effect concentration (EC50) or lowest effect concentration (LEC), yielded a significant relationship between both methods described by the equation log(BaPIEF(EC50) = 0.55 x log(BaPIEF(LEC)) + 0.07 (r2 = 0.913). BaP-IEFs as derived from our measurements and as reported in literature and measured in other in vitro assays deviated up to a factor of 17 among the different studies, but the potency rankings were comparable. For the PAH mixture as on average present in the human diet an overall tetrachlorodibenzo-p-dioxin (TCDD)-IEF of 1 x 10(-4) was estimated. The total PAH based TCDD induction equivalents (IEQ) intake then was calculated 300 pg/day, which is approximately 2 times higher then the PHAH based TCDD-EQ intake reported for humans.

Effects of an extract of oystercatcher (Haematopus ostralegus) eggs from the Zeehavenkanaal in The Netherlands, and of its major contaminant, hexachlorobenzene, on the chicken embryo.

Oystercatchers (Haematopus ostralegus) foraging on the canal 'Zeehavenkanaal' in the Netherlands have been shown to accumulate appreciable amounts of contaminants, especially hexachlorobenzene. The present study was performed to assess the embryotoxic effects of the present contaminants. To this end, a two step approach was followed. In step one, the toxic effects of hexachlorobenzene were studied in the chicken embryo bioassay, using concentrations realistic for the field situation. In step two, yolks of oystercatcher eggs were extracted and the embryotoxic potency of this extract was studied in the same bioassay, using doses of 1, 10 and 100% of the contaminant load in one average egg. The extract contained hexachlorobenzene and PCBs. However, presence of other compounds could not be excluded, since these were not analysed. Hexachlorobenzene induced a nonsignificant decrease in lymphocyte density in the bursa of Fabricius. The egg extract caused a 3.5 fold induction of EROD activity at the highest dose applied, and decreased lymphocyte density in the bursa of Fabricius.

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.