A prospective whole-mixture approach to assess risk of the food and chemical exposome.

Many widely used chemicals result in ubiquitous human exposure from multiple sources, including diet. Legislation mainly deals with the toxicological evaluation of single substances owing to a methodological and conceptual lack of alternatives, and does so within defined silos subject to over 40 distinct regulations in the EU alone. Furthermore, much of the research and many of the initiatives concerned with the assessment and evaluation of chemical mixtures and their potential effects on human health rely on retrospective analysis. Here we propose an approach for the prospective identification, assessment and regulation of mixtures relevant to human health. We address two distinct aspects of toxicology-which chemicals actually do occur together, and how potential mixture-related health hazards can be predicted-with an adapted concept of the exposome and large-scale hazard screens. The proactive use of the likelihood of co-exposure, together with the new approach of methods-based testing, may be a timely and feasible way of identifying those substances and mixtures where hazards may have been overlooked and regulatory action is needed. Ideally, we would generate co-exposure patterns for specific consumer groups, depending on lifestyle and dietary habits, to assess the specific risk of identified mixtures.

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone ß-subunit (lhß) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone ß-subunit (fshß) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshß in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes.

Nutritional status and gene expression along the somatotropic axis in roach (Rutilus rutilus) infected with the tapeworm Ligula intestinalis.

The tapeworm Ligula intestinalis inhibits gametogenesis of its fish host, the roach (Rutilus rutilus). We investigated whether L. intestinalis infection makes significant demands on nutritional resources and consequently manipulates the endocrine somatotropic axis of roach. Two groups of naturally infected and uninfected roach were studied: a field group (natural feeding) and a laboratory group (ad libitum food supply). In females, no significant impact of parasitization on storage substrates (glycogen, lipids, and protein) was detected, whereas in males, either lipid content of the liver (field group) or lipid of the muscle and glycogen of the liver (laboratory group) were slightly decreased. Except for the females of the field group, higher mRNA expression of growth hormone (gh) in the pituitary of infected fish was observed. Furthermore, the expression of hypophyseal somatolactin α and ß (slα, slß) was up-regulated in infected females of the field and laboratory group, respectively. In liver and muscle, mRNA expression of insulin-like growth factors (igf1, igf2) and igf receptor (igfr) remained either unchanged or were up-regulated with infection. Parasitization showed inconsistent effects on gh receptor 1 (ghr1) expression in liver and muscle, whereas ghr2 mRNA was mostly not influenced by infection. In general, the expression profile of genes involved in the somatotropic axis as well as the content of storage substances in infected roach did not resemble that of food-deprived fish either under natural or ad libitum feeding. In conclusion, the present study does not indicate starvation of L. intestinalis infected roach, and it is suggested that the inhibition of reproduction attenuated the nutritional demand of parasitization.

Influence of temperature on puberty and maturation of pikeperch, Sander lucioperca.

Among external factors, temperature is known to exhibit a prominent role in reproduction of temperate fish species. Here, temperature related induction of puberty in pikeperch Sander lucioperca was investigated. For the first time the key factors of the pikeperch brain-pituitary-gonad axis, targeting the mRNA expression of the luteinising hormone (LH) and the follicle stimulating hormone (FSH), as well as the plasma sex steroids estradiol (E2), testosterone (T), 11-ketotestosteron (11-KT) and 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) were addressed in the experiment. Concomitant the maturational stages were described histologically. After 3 months, female pikeperch kept at 12°C revealed significant increases in the GSI and plasma E2 concentration and 90% of the females were mid-vitellogenic. After 5 months, females kept between 9 and 15°C exhibited significant up-regulation of E2 and GSI as well as comparable histological outcome. At 6 and 23°C in nearly all females stagnation of oogenesis was recorded. Congruently, T was increased at 12 and 15°C. Expression analysis revealed a significant up-regulation of LHß and FSHß mRNA in females from early-vitellogenesis, and from mid-spermatogenesis in males, correlated to elevated plasma concentrations of steroids (except for E2 in males). In conclusion, moderate temperatures (12-15°C for) for at least 3 months were required to proceed with first maturation in juvenile pikeperch. The most efficient effect was observed at 12°C, while high (23°C) or low (6°C) temperatures prevented gonadal maturation. So temperature was identified as a prime factor in the induction of puberty in pikeperch, as revealed by histological as well as endocrine parameters.