Ingvar Brandt

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.

Health Effects of Endocrine-Disrupting Chemicals on Wildlife, with Special Reference to the European Situation

Many wildlife species may be exposed to biologically active concentrations of endocrine-disrupting chemicals. There is strong evidence obtained from laboratory studies showing the potential of several environmental chemicals to cause endocrine disruption at environmentally realistic exposure levels. In wildlife populations, associations have been reported between reproductive and developmental effects and endocrine-disrupting chemicals. In the aquatic environment, effects have been observed in mammals, birds, reptiles, fish, and mollusks from Europe, North America, and other areas. The observed abnormalities vary from subtle changes to permanent alterations, including disturbed sex differentiation with feminized or masculinized sex organs, changed sexual behavior, and altered immune function. For most reported effects in wildlife, however, the evidence for a causal link with endocrine disruption is weak or nonexist-ing. Crucial in establishing causal evidence for chemical-induced wildlife effects appeared semifield or laboratory studies using the wildlife species of concern. Impaired reproduction and development causally linked to endocrine-disrupting chemicals are well documented in a number of species and have resulted in local or regional population changes. These include: • Masculinization (imposex) in female marine snails by tributyltin, a biocide used in antifouling paints, is probably the clearest case of endocrine disruption caused by an environmental chemical. The dogwhelk is particularly sensitive, and imposex has resulted in decline or extinction of local populations worldwide, including coastal areas all over Europe and the open North Sea. • DDE-induced egg-shell thinning in birds has caused severe population declines in a number of raptor species in Europe and North America. • Endocrine-disrupting chemicals have adversely affected a variety of fish species. In the vicinity of certain sources (e.g., effluents of water treatment plants) and in the most contami nated areas is this exposure causally linked with the effects on reproductive organs that could have implications for fish populations. However, there is also a more widespread occurrence of endocrine disruption in fish in the U.K., where estrogenic effects have been demonstrated in freshwater systems, in estuaries, and in coastal areas. • In mammals, the best evidence comes from the field studies on Baltic gray and ringed seals, and from the Dutch semifield studies on harbor seals, where both reproduction and immune functions have been impaired by PCBs in the food chain. Reproduction effects resulted in population declines, whereas impaired immune function has likely contributed to the mass mortalities due to morbillivirus infections. • Distorted sex organ development and function in alligators has been related to a major pesticide spill into a lake in Florida, U.S.A. The observed estrogenic/antiandrogenic effects in this reptile have been causally linked in experimental studies with alligator eggs to the DDT complex. Although most observed effects currently reported concern heavily polluted areas, endocrine disruption is a potential global problem. This is exemplified by the widespread occurrence of imposex in marine snails and the recent findings of high levels of persistent potential endocrine-disrupting chemicals in several marine mammalian species inhabiting oceanic waters.

Controlling persistent organic pollutants-what next?

Within the context of current international initiatives on the control of persistent organic pollutants (POPs), an overview is given of the scientific knowledge relating to POP sources, emissions, transport, fate and effects. At the regional scale, improvements in mass balance models for well-characterised POPs are resulting in an ability to estimate their environmental concentrations with sufficient accuracy to be of help for some regulatory purposes. The relevance of the parameters used to define POPs within these international initiatives is considered with an emphasis on mechanisms for adding new substances to the initial lists. A tiered approach is proposed for screening the large number of untested chemical substances according to their long-range transport potential, persistence and bioaccumulative potential prior to more detailed risk assessments. The importance of testing candidate POPs for chronic toxicity (i.e. for immunotoxicity, endocrine disruption and carcinogenicity) is emphasised as is a need for the further development of relevant SAR (structure activity relationship) models and in vitro and in vivo tests for these effects. Where there is a high level of uncertainty at the risk assessment stage, decision-makers may have to rely on expert judgement and weight-of-evidence, taking into account the precautionary principle and the views of relevant stake-holders. Close co-operation between the various international initiatives on POPs will be required to ensure that assessment criteria and procedures are as compatible as possible.

The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ development.

Many environmental contaminants are known or suspected to interfere with hormonal function in animals. In vivo test methods to detect and characterize chemicals that disrupt the endocrine system are therefore urgently needed. In this study, we assessed the usefulness of abnormalities of the reproductive organs as test endpoints for estrogenic activity of xenobiotics in Japanese quail embryos. Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), were injected into the yolks of embryonated eggs. At a dose as low as 2 ng EE2/g egg, all male embryos became feminized, containing ovary-like tissue in the left testis. The extent of feminization of the testes was determined by measuring the relative area of the ovary-like component. Persistent Müllerian ducts (oviducts) in male embryos, and malformations of the Müllerian ducts in females occurred at 2 ng EE2/g egg and higher doses. DES was approximately one-third to one-tenth as potent as EE2. The morphological changes studied were dose-dependent, indicating that they are useful as test endpoints for estrogenic activity. Feminization of the left testis in males proved to be the most sensitive endpoint. We propose the quail egg as a simple in vivo test system for estrogenic compounds.

Accumulation of methylsulfonyl derivatives of some bronchial-seeking polychlorinated biphenyls in the respiratory tract of mice

Abstract Seventeen polychlorobiphenyls (PCBs), 14 C-labeled and/or unlabeled, were studied with regard to their deposition as such or as metabolites in the lung tissues after iv or ip injection in mice. Four of these compounds were known by autoradiography to possess affinity for bronchial tissues. Lung extracts of mice treated with the 11 labeled PCBs were analyzed in a partition scheme to indicate the presence of hydroxylated and/or methylsulfonyl metabolites. Extracts of lungs from mice treated with the unlabeled compounds were analyzed by gas chromatography. The extracts that appeared to contain methylsulfonyl metabolites were further analyzed by mass fragmentography. Five of the biphenyls studied were each found to appear in the lungs as two isomeric methylsulfonyl PCBs. The structures of the major metabolites were confirmed by comparison with synthetic reference compounds. One of the biphenyls previously known to accumulate in the bronchi was found not to occur in the lung as methylsulfonyl derivatives, but most probably only as the unchanged compound. None of the remaining 11 PCBs studied was found to occur as methylsulfonyl derivatives in the lung.

The Synthetic Progestin Levonorgestrel Is a Potent Androgen in the Three-Spined Stickleback (Gasterosteus aculeatus)

The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L(-1) concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (Gasterosteus aculeatus) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L(-1) for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L(-1) including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity.

The distribution of 2,21,3,4,41,61, and 2,31, 4,41,51,6- hexachlorobiphenyl in mice studied by whole-body autoradiography.

The distribution of 14C-labelled hexachlorobiphenyl in male and pregnant female mice was studied by whole-body autoradiography and liquid scintillation counting. Shortly after an intravenous injection there was a strong uptake of polychlorinated biphenyl (PCB) in the corpora lutea, the adrenal cortex and the liver and radioactivity was retained fro a long time in these tissues. Even in the fetal adrenal cortex a considerable accumulation occurred. High activity was also present in the brown fat, the lung, the the myocardium, the gastric mucosa, the pituitary and the bone marrow. Radioactivity penetrated slowly to the brain and fetal tissues but the concentration remained consistently low. In the adipose tissues there was a gradual increase in radioactivity and the autoradiograms from the longer survival times were dominated by these sites. There was a continuous biliary excretion of labelled material to the small intestine; this occurred in the fetuses as well.

Methods for studying xenoestrogenic effects in birds

The embryonated bird egg provides a simple whole organism test system that allows examination of xenoestrogenic effects at different levels of biological organisation. Test compounds are injected into the yolk, the albumen or the air chamber at defined stages of embryonic development. Bioavailability and embryonic exposure may be determined by autoradiography and image analysis. Females represent the heterogametic sex (ZW) and estrogens determine differentiation into the female phenotype in birds. Xenoestrogenic effects can be examined by markers of gene expression and anatomical or histological characterization of the gonads and tubular sex organs. Chicks may be raised to sexual maturity and examination of sexual behaviour and reproductive physiology performed. The Japanese quail is a suitable test organism due to its small size and early sexual maturation.

The zebrafish gill model: induction of CYP1A, EROD and PAH adduct formation.

Zebrafish (Danio rerio) is a common model species in fish toxicology, and the zebrafish gill is potentially useful in screening waterborne pollutants. We have previously developed a gill-based ethoxyresorufin-O-deethylase (EROD) assay, and proposed gill EROD activity as a biomarker for exposure to waterborne aryl hydrocarbon receptor (AHR) agonists. In this study we modified the gill EROD assay for use in zebrafish. We used immunohistochemistry to localize CYP1A induction, and microautoradiography to localize irreversible binding of the prototypic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA) in zebrafish gills. Gill filament and liver microsomal EROD activities were measured after waterborne exposure of zebrafish and rainbow trout to benzo[a]pyrene (BaP) or beta-naphthoflavone (betaNF). The results showed considerably lower relative EROD induction by betaNF (1microM) in zebrafish than in rainbow trout, both in gills (13-fold versus 230-fold compared to control) and in liver (5-fold versus 320-fold compared to control). The induced hepatic EROD activity was similar in the two species, whereas the basal activity was considerably higher in zebrafish than in rainbow trout. In zebrafish gills, betaNF enhanced DMBA adduct formation and CYP1A immunostaining. Ellipticine blocked DMBA adduct formation and EROD activity following betaNF exposure but had no effect on CYP1A immunostaining. A notable finding was that the localization of DMBA adducts differed from that of CYP1A protein in betaNF-induced fish; CYP1A immunoreactivity was evenly distributed in the gills whereas DMBA adduction was confined to the leading edges of the filaments and the gill rakers, i.e., structures being highly exposed to DMBA-containing inhaled water. The results show that the modified method is suitable for determination of gill EROD activity in zebrafish, although rainbow trout seems more sensitive. They also imply that the sites of DMBA adduct formation in zebrafish gills are markedly influenced by kinetic factors.

A path forward in the debate over health impacts of endocrine disrupting chemicals

Several recent publications reflect debate on the issue of “endocrine disrupting chemicals” (EDCs), indicating that two seemingly mutually exclusive perspectives are being articulated separately and independently. Considering this, a group of scientists with expertise in basic science, medicine and risk assessment reviewed the various aspects of the debate to identify the most significant areas of dispute and to propose a path forward. We identified four areas of debate. The first is about the definitions for terms such as “endocrine disrupting chemical”, “adverse effects”, and “endocrine system”. The second is focused on elements of hormone action including “potency”, “endpoints”, “timing”, “dose” and “thresholds”. The third addresses the information needed to establish sufficient evidence of harm. Finally, the fourth focuses on the need to develop and the characteristics of transparent, systematic methods to review the EDC literature. Herein we identify areas of general consensus and propose resolutions for these four areas that would allow the field to move beyond the current and, in our opinion, ineffective debate.