Leif Norrgren

Science and policy on endocrine disrupters must not be mixed: a reply to a “common sense” intervention by toxicology journal editors

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone.

In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.

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

To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish (Danio rerio) were exposed to 17α-ethinylestradiol (actual concentration 15.4±1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10–20 dph; 20–30 dph; 20–40 dph; 20–60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4±1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.

Differences in uptake of inorganic mercury and cadmium in the gills of the zebrafish, Brachydanio rerio

The influence of calcium and calcium channel blockers on the apical uptake of cadmium and inorganic mercury was studied in the branchial epithelium of the zebrafish (Brachydanio rerio). Autoradiography was used to determine the distribution of Cd and Hg after uptake in the epithelium. Zebrafish were exposed to 20 nM 109Cd or 203Hg for 15–30 min. Cd uptake was considerably lower at 2 mM Ca2+ than at 0.2 mM Ca2+, whereas uptake of Hg was not influenced. The calcium-channel blocker verapamil caused a concentration-dependent decrease in uptake of Cd. Uptake of Hg increased when fish were exposed to 150 μM verapamil, but was not affected by 250 μM verapamil. Fish exposed to Cd or Hg at 0.2 mM Ca2+ and in the presence of 1 μM lanthanum (La3+) showed a lower branchial uptake of the metals than the controls. In another experiment fish were exposed to 109Cd or 203Hg (10 nM) for 24 h. Autoradiograms of the gills indicated a high Cd uptake in some epithelial cells of the primary filament. Identifiable 109Cd-positive cells had the appearance of chloride cells. Hg was relatively evenly distributed between the primary filament and the secondary lamella. No specific type of cell with a high uptake of Hg was detected. The results show that there are differences in apical uptake of Cd and Hg, probably due to speciation differences between Cd and Hg in fresh water.

Leucocyte adhesion protein deficiency in Irish setter dogs

Investigation of 12 Irish setter puppies from six litters with severe recurrent infections, neutrophilia and low body weight revealed a leucocyte adhesion protein deficiency with a total lack of CD11b and CD18. Their neutrophil function was severely impaired with a totally absent capacity to ingest C3b-opsonized particles, a significantly impaired capacity to ingest IgG-opsonized particles and significantly diminished adherence to nylon wool when compared with neutrophils from healthy control dogs. The chemiluminescence of patient neutrophils activated by C3b-opsonized particles was, consequently, significantly decreased compared with that of control neutrophils, while the respiratory burst assayed by phorbolmyristate acid (PMA) stimulated nitroblue tetrazolium (NBT)-reduction was normal in the patient group. Random migration and chemotactic responses of patient and control neutrophils, were similar. The etiology, pathogenesis and clinical manifestations of the Irish setter leucocyte adhesion deficiency were similar to that of the leucocyte adhesion deficiency in humans.

Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the three-spined stickleback, Gasterosteus aculeatus

Abstract Female three-spined sticklebacks were fed with freeze-dried chironomids contaminated with low or high doses of polybrominated diphenyl ethers (PBDEs) (Bromkal 70-5DE), polychlorinated naphthalenes (PCNs) (Halowax 1014) and polychlorinated biphenyls (PCBs) (Clophen A50). After 3.5 months of exposure, reproduction studies were started. No significant difference in number of eggs laid was found between the control and exposed groups. Spawning success (considered successful if it occurred within 24 h) in the control group was 80%, whereas it was 20% and 25% in the groups that received high doses of Bromkal 70-5DE or Clophen A50, respectively. After spawning, the fish were dissected for chemical, biochemical and morphological analyses. Levels of PBDE in the low- and high-dose Bromkal 70-5DE groups were 861 ± 271 and 1630 ± 275 mg/kg fat, respectively, whereas the corresponding concentrations of PCN in the Halowax 1014 groups were 845 ± 43 and 1929 ± 72 mg/kg fat, respectively. Concentrations of PCB in fish from the Clophen A50 groups were 1972 ± 158 and 3594 ± 521 mg/kg fat, respectively. Uptake efficiency was approx. 40% for PCB and 20% for PBDE and PCN. Hepatic cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD) activity was induced by Clophen A50 and Halowax 1014, whereas Bromkal 70-5DE did not significantly induce the enzyme system. EROD activity was 0.8 pmol/mg prot./min in the control group, whereas it was between 2.7 and 16.3 pmol/mg prot./min in the exposed groups. Furthermore, the morphological examination of the liver disclosed pronounced lipid accumulation in all exposed groups.

Toxicity of 15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos.

Extensive use of veterinary pharmaceuticals may result in contamination of water bodies adjacent to pasture land or areas where animal manure has been applied. In order to evaluate the potential risk to fish embryos 15 veterinary pharmaceuticals were investigated by use of an extended zebrafish embryo toxicity test. Chemical analysis of the exposure medium was performed by solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for 11 of the compounds and potential metabolism by the embryos was studied for albendazole, febantel, fenbendazole and oxfendazole. Newly fertilized zebrafish eggs were exposed under static conditions in 96-well plates for 6 days to the pharmaceuticals: 5 antibacterials and 10 antiparasitics. Endpoints including mortality, malformations and other sublethal responses were recorded at 24, 48 and 144 h post fertilization (hpf). The pharmaceuticals causing the highest toxicity were antiparasitics whereas the tested antibacterials, danofloxacin, enrofloxacin, tylosine, trimethoprim and oxytetracyclin had a much lower toxic potency in zebrafish embryos. Most toxic were fenbendazole, albendazole and flumethrin with no observed effect concentrations (NOECs) around 0.02 mg/L. The overall NOEC was determined by lethality for the following pharmaceuticals: albendazole, fenbendazole and oxfendazole. Sublethal endpoints, including malformations, side-laying embryos, tremors, reduced movements and altered heart rate increased the sensitivity of the tests and determined the overall NOECs for febantel, doramectin, ivermectin, flumethrin and toltrazuril. Exposure to doramectin and ivermectin caused a decrease in movements at 24 hpf and a decrease in heart rate at 48 hpf. Flumethrin exposure resulted in decreased time to hatching, except at the highest concentrations, and caused an increase in heart rate at 48 hpf. In contrast, toltrazuril caused an increased time to hatching and a decrease in heart rate. Chemical analysis of the exposure medium after the tests revealed great differences between nominal and measured concentrations, emphasizing the need of including analysis of the actual exposure concentrations. The results indicated that metabolism of albendazole into its sulfoxide protected the embryos from toxicity. Albendazole was metabolized efficiently into albendazole sulfoxide at lower exposure concentrations, resulting in reduced toxicity. At higher concentrations, an increasing proportion of albendazole remained unmetabolized and embryo mortality occurred. Metabolism by the embryos of febantel into fenbendazole and oxfendazole and of fenbendazole into oxfendazole was demonstrated. It is suggested that the toxic effect of febantel in zebrafish embryos is due to metabolism into fenbendazole.

Liver morphology and cytochrome P450 activity in fry of rainbow trout after microinjection of lipid-soluble xenobiotics in the yolk-sac embryos

Abstract In this experimental study a microinjection technique for the administration of lipid-soluble xenobiotics in rainbow trout embryos was applied. One week prior to hatching, rainbow trout embryos were injected in the yolk-sac with commercial blends of polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), polychlorinated paraffins (PCPs) and polybrominated diphenyl ethers (PBDEs). Six weeks later, the morphology of the liver was examined, and the hepatic cytochrome P450-dependent ethoxyresorufin- O -deethylase (EROD) activity was determined in the swim-up fry. A dose-dependent induction of the EROD activity was found for the PCBs and the PCNs, indicating that application of the microinjection technique in fish embryo bioassays, combined with studies of liver morphology and biochemistry, facilitate detection of biological responses to certain lipid-soluble substances.

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).

Gender-specific proteomic responses in zebrafish liver following exposure to a selected mixture of brominated flame retardants.

Proteomic effect screening in zebrafish liver was performed to generate hypotheses following exposure (21 days) to a structurally diverse mixture of brominated flame retardants (BFRs). Fish were exposed to two doses (10 and 100 nmol/g feed). Two-dimensional gel-electrophoresis, image analysis and MALDI-TOF mass-spectrometry revealed 13 and 19 significant responses in males and females, respectively. Effects on proteins related to cellular maintenance and stress were observed in both genders. Regulated proteins were gender-specific, but functionally indicated common protective responses (peroxiredoxin 6 and Zgc:92891 in males and transketolase in females) suggesting oxidative stress. Betaine homocysteine methyltransferase (BHMT) was induced in both genders. In addition a female-specific downregulation of ironhomeostatic proteins (iron-regulatory protein 1 and transferrin) were observed. Our proteomic approach revealed novel responses that suggest important gender-specific sensitivity to BFRs that should be considered when interpreting adverse effects of BFRs.