Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test.

BACKGROUND: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). OBJECTIVE: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. METHODS: Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. RESULTS: Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.

Combined chronic dietary exposure to four nephrotoxic metals exceeds tolerable intake levels in the adult population of 10 European countries.

A mixture risk assessment (MRA) for four metals relevant to chronic kidney disease (CKD) was performed. Dietary exposure to cadmium or lead alone exceeded the respective reference values in the majority of the 10 European countries included in our study. When the dietary exposure to those metals and inorganic mercury and inorganic arsenic was combined following a classical or personalised modified reference point index (mRPI) approach, not only high exposure (95th percentile) estimates but also the mean exceeded the tolerable intake of the mixture in all countries studied. Cadmium and lead contributed most to the combined exposure, followed by inorganic arsenic and inorganic mercury. The use of conversion factors for inorganic arsenic and inorganic mercury from total arsenic and total mercury concentration data was a source of uncertainty. Other uncertainties were related to the use of different principles to derive reference points. Yet, MRA at the target organ level, as performed in our study, could be used as a way to efficiently prioritise assessment groups for higher-tier MRA. Since the combined exposure to the four metals exceeded the tolerable intake, we recommend a refined MRA based on a common, specific nephrotoxic effect and relative potency factors (RPFs) based on a similar effect size.

Prioritization of chemicals in food for risk assessment by integrating exposure estimates and new approach methodologies: A next generation risk assessment case study.

Next generation risk assessment is defined as a knowledge-driven system that allows for cost-efficient assessment of human health risk related to chemical exposure, without animal experimentation. One of the key features of next generation risk assessment is to facilitate prioritization of chemical substances that need a more extensive toxicological evaluation, in order to address the need to assess an increasing number of substances. In this case study focusing on chemicals in food, we explored how exposure data combined with the Threshold of Toxicological Concern (TTC) concept could be used to prioritize chemicals, both for existing substances and new substances entering the market. Using a database of existing chemicals relevant for dietary exposure we calculated exposure estimates, followed by application of the TTC concept to identify substances of higher concern. Subsequently, a selected set of these priority substances was screened for toxicological potential using high-throughput screening (HTS) approaches. Remarkably, this approach resulted in alerts for a selection of substances that are already on the market and represent relevant exposure in consumers. Taken together, the case study provides proof-of-principle for the approach taken to identify substances of concern, and this approach can therefore be considered a supportive element to a next generation risk assessment strategy.

A Case Study with Triazole Fungicides to Explore Practical Application of Next-Generation Hazard Assessment Methods for Human Health.

The ongoing developments in chemical risk assessment have led to new concepts building on integration of sophisticated nonanimal models for hazard characterization. Here we explore a pragmatic approach for implementing such concepts, using a case study of three triazole fungicides, namely, flusilazole, propiconazole, and cyproconazole. The strategy applied starts with evaluating the overall level of concern by comparing exposure estimates to toxicological potential, followed by a combination of in silico tools and literature-derived high-throughput screening assays and computational elaborations to obtain insight into potential toxicological mechanisms and targets in the organism. Additionally, some targeted in vitro tests were evaluated for their utility to confirm suspected mechanisms of toxicity and to generate points of departure. Toxicological mechanisms instead of the current "end point-by-end point" approach should guide the selection of methods and assays that constitute a toolbox for next-generation risk assessment. Comparison of the obtained in silico and in vitro results with data from traditional in vivo testing revealed that, overall, nonanimal methods for hazard identification can produce adequate qualitative hazard information for risk assessment. Follow-up studies are needed to further refine the proposed approach, including the composition of the toolbox, toxicokinetics models, and models for exposure assessment.

Consumption of plant food supplements in the Netherlands.

The use of food supplements containing herbs or other botanical ingredients (plant food supplements, PFS) is on the rise. In some cases, PFS can contain compounds that are toxic and may pose a health risk. To assess the potential health risks, information on the consumption of PFS is required, however, this was lacking for the Netherlands. In the current study, the consumption of PFS was investigated for several subgroups in the Dutch population, including children. Data from the Dutch National Food Consumption Surveys were used to get a first impression on the consumption of PFS. To obtain more detailed information, a specific PFS consumption survey was performed using online questionnaires. First, a screening survey was performed among a representative sample of 75 100 adults and children of the Dutch population, followed by a main survey among 739 selected PFS users in eight different age and gender subgroups. The prevalence of PFS users in the Dutch population was approximately 10% for men, 17% for women and 13% for children. A wide variety of PFS was used, with around 600 different PFS reported, containing 345 different botanicals. The most frequently used botanicals were echinacea (Echinacea purpurea), ginkgo (Ginkgo biloba), cranberry (Vaccinium macrocarpon), ginseng (Panax ginseng) and algae (such as species belonging to the genus Spirulina or Chlorella). Because PFS are widely used in the Dutch population, it is important to evaluate the potential risks associated with PFS consumption in the Netherlands, including potential herb-drug interactions. The data collected in this study are of great value to assess these risks.

Genetic variation in vitamin B-12 content of bovine milk and its association with SNP along the bovine genome.

Vitamin B-12 (also called cobalamin) is essential for human health and current intake levels of vitamin B-12 are considered to be too low. Natural enrichment of the vitamin B-12 content in milk, an important dietary source of vitamin B-12, may help to increase vitamin B-12 intake. Natural enrichment of the milk vitamin B-12 content could be achieved through genetic selection, provided there is genetic variation between cows with respect to the vitamin B-12 content in their milk. A substantial amount of genetic variation in vitamin B-12 content was detected among raw milk samples of 544 first-lactation Dutch Holstein Friesian cows. The presence of genetic variation between animals in vitamin B-12 content in milk indicates that the genotype of the cow affects the amount of vitamin B-12 that ends up in her milk and, consequently, that the average milk vitamin B-12 content of the cow population can be increased by genetic selection. A genome-wide association study revealed significant association between 68 SNP and vitamin B-12 content in raw milk of 487 first-lactation Dutch Holstein Friesian cows. This knowledge facilitates genetic selection for milk vitamin B-12 content. It also contributes to the understanding of the biological mechanism responsible for the observed genetic variation in vitamin B-12 content in milk. None of the 68 significantly associated SNP were in or near known candidate genes involved in transport of vitamin B-12 through the gastrointestinal tract, uptake by ileum epithelial cells, export from ileal cells, transport through the blood, uptake from the blood, intracellular processing, or reabsorption by the kidneys. Probably, associations relate to genes involved in alternative pathways of well-studied processes or to genes involved in less well-studied processes such as ruminal production of vitamin B-12 or secretion of vitamin B-12 by the mammary gland.

Sweet buttermilk intake reduces colonisation and translocation of Listeria monocytogenes in rats by inhibiting mucosal pathogen adherence.

The bovine milk fat globule membrane (MFGM) contains several antimicrobial components with proven efficacy in vitro, but in vivo evidence is scarce. The present study was performed to determine the efficacy of the bovine MFGM in vivo. Rats were fed diets based on bovine skimmed milk powder (low in MFGM) or bovine sweet buttermilk powder (high in MFGM). After dietary adaptation, rats were orally infected with Salmonella enteritidis or Listeria monocytogenes. Whereas sweet buttermilk powder did not protect rats against infection with S. enteritidis, it protected against L. monocytogenes, as shown by a lower colonisation and translocation of this pathogen. Protection coincided with higher listericidal capacity of gastric and caecal contents. The digestion products of phosphoglycerides and sphingomyelin are bactericidal in vitro. To study their role, rats were fed diets containing either 0·1 % phosphatidylcholine or sphingomyelin, or a control diet. After dietary adaptation, rats were infected with L. monocytogenes. Since Listeria colonisation was not affected by these diets, phosphoglycerides and sphingomyelin are not involved in the protective effect of sweet buttermilk. Additional in vitro experiments were performed to further explore the mechanism of the beneficial effects of sweet buttermilk. Inhibition of the adherence of L. monocytogenes to the intestinal mucosa is the most likely explanation, since sweet buttermilk powder inhibited the binding of L. monocytogenes in both a haemagglutination assay and a Caco-2 cell adherence assay. In conclusion, sweet buttermilk powder, which is rich in MFGM, protects against L. monocytogenes infection in rats, probably by preventing adherence of this pathogen to the intestinal mucosa.

Dietary calcium phosphate promotes Listeria monocytogenes colonization and translocation in rats fed diets containing corn oil but not milk fat.

Most Gram-positive bacteria are susceptible to the bactericidal action of fatty acids and bile acids. Because dietary calcium phosphate (CaP(i)) lowers the intestinal concentration of these antimicrobial agents, high CaP(i) intake may enhance intestinal colonization of Gram-positive pathogens and the subsequent pathogenesis. In this study, we tested this hypothesis in a rat model using Listeria monocytogenes. Rats were fed diets containing low (20 micromol/g diet) or high (160 micromol/g diet) amounts of CaP(i). Dietary fat was provided as corn oil or milk fat. Rats were orally inoculated with L. monocytogenes. When rats consumed diets containing corn oil, high CaP(i) intake indeed stimulated colonization of L. monocytogenes and increased L. monocytogenes translocation and diarrhea. In addition, supplemental CaP(i) enhanced ex vivo growth of L. monocytogenes in fecal extracts of rats fed corn oil diets, suggesting that high CaP(i) intake decreased a luminal inhibitory factor. The concentrations of bile salts and fatty acids, which were highly listericidal in vitro, were indeed considerably decreased in fecal water of rats in the high calcium corn oil group. Surprisingly, dietary CaP(i) did not affect colonization and translocation of L. monocytogenes in rats fed the milk fat diet, nor did CaP(i) enhance ex vivo growth in fecal extracts. This absence of Listeria stimulation was associated with a lack of effect of dietary CaP(i) on fecal soluble fatty acids. In addition, residual soluble bile salts were higher in rats fed the high CaP(i) milk fat diet compared with the high CaP(i) corn oil diet. These results suggest that the stimulating effect of CaP(i) on L. monocytogenes infection depends on the type of dietary fat consumed.