Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A. Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3',4,5'-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 μM and inhibited migration with 37.5 ± 14.8% at 3 μM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 μM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 μM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 μM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 μM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells. TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.

Dietary exposure to nitrate and nitrite occurs via three main sources; occurrence in (vegetable) foods, food additives in certain processed foods and contaminants in drinking water. While nitrate can be converted to nitrite in the human body, their risk assessment is usually based on single substance exposure in different regulatory frameworks. Here, we assessed the long-term combined exposure to nitrate and nitrite from food and drinking water. Dutch monitoring data (2012–2018) and EFSA data from 2017 were used for concentration data. These were combined with data from the Dutch food consumption survey (2012–2016) to assess exposure. A conversion factor (median 0.023; range 0.008–0.07) was used to express the nitrate exposure in nitrite equivalents which was added to the nitrite exposure. The uncertainty around the conversion factor was taken into account by using conversion factors randomly sampled from the abovementioned range. The combined dietary exposure was calculated for the Dutch population (1–79 years) with different exposure scenarios to address regional differences in nitrate and nitrite concentrations in drinking water. All scenarios resulted in a combined exposure above the acceptable daily intake for nitrite ion (70 µg/kg bw), with the mean exposure varying between 95–114 µg nitrite/kg bw/day in the different scenarios. Of all ages, the combined exposure was highest in children aged 1 year with an average of 250 µg nitrite/kg bw/day. Vegetables contributed most to the combined exposure in food in all scenarios, varying from 34%–41%. Food additive use contributed 8%–9% to the exposure and drinking water contributed 3%–19%. Our study is the first to perform a combined dietary exposure assessment of nitrate and nitrite while accounting for the uncertain conversion factor. Such a combined exposure assessment overarching different regulatory frameworks and using different scenarios for drinking water is a better instrument for protecting human health than single substance exposure. GRAPHICAL ABSTRACT

Humans are exposed to mycotoxins on a regular basis. Exposure to a mixture of mycotoxins may, therefore, result in a combination of adverse effects, or trigger the same effects. This should be accounted for when assessing the combined risk of multiple mycotoxins. Here, we show the outcome of using different approaches in assessing the risks related to the combined exposure to mycotoxins. We performed a tiered approach using assessment groups with a common target organ (kidney, liver and haematologic system), or a common adverse effect (phenomenon) (reduced white blood cell count), to combine the exposure to mycotoxins. The combined exposure was calculated for the individuals in this assessment, using the Monte Carlo Risk Assessment (MCRA) tool. The risk related to this combined exposure was assessed using toxicological reference values, e.g., health based guidance values. We show that estimating the combined risk by adding the single compounds’ risk distributions slightly overestimates the combined risk in the 95th percentile, as compared to combining the exposures at an individual level. We also show that relative potency factors can be used to refine the mixture risk assessment, as compared to ratios of toxicological reference values with different effect sizes and assessment factors.

Mycotoxins are natural metabolites produced by fungi that contaminate food and feed worldwide. They can pose a threat to human and animal health, mainly causing chronic effects, e.g., immunotoxic and carcinogenic. Due to climate change, an increase in European population exposure to mycotoxins is expected to occur, raising public health concerns. This urges us to assess the current human exposure to mycotoxins in Europe to allow monitoring exposure and prevent future health impacts. The mycotoxins deoxynivalenol (DON) and fumonisin B₁ (FB₁) were considered as priority substances to be studied within the European Human Biomonitoring Initiative (HBM4EU) to generate knowledge on internal exposure and their potential health impacts. Several policy questions were addressed concerning hazard characterization, exposure and risk assessment. The present article presents the current advances attained under the HBM4EU, research needs and gaps. Overall, the knowledge on the European population risk from exposure to DON was improved by using new harmonised data and a newly derived reference value. In addition, mechanistic information on FB₁ was, for the first time, organized into an adverse outcome pathway for a congenital anomaly. It is expected that this knowledge will support policy making and contribute to driving new Human Biomonitoring (HBM) studies on mycotoxin exposure in Europe.

Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure–health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) ‘reduced body weight gain’, we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) ‘inhibition of ceramide synthases’ leading to the AO ‘neural tube defects’. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure–health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM.

Human and rat reproductive systems differ significantly with respect to hormonal cyclicity and endometrial cell behavior. However, species-differences in endometrial cell responses upon hormonal stimulation and exposure to potentially toxic compounds are poorly characterized. In this study, human and rat endometrial hormonal responses were assessed in vitro using a 3D co-culture model of primary human and rat endometrial cells. The models were exposed to the aryl hydrocarbon receptor (AHR) ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), laquinimod, and its AHR active metabolite DELAQ. In both the human and rat endometrial models, estrogen receptor and progesterone receptor gene expression was modulated by the hormonal treatments, comparable to the in vivo situation. AHR gene expression in the human endometrial model did not change when exposed to hormones. In contrast, AHR expression decreased 2-fold in the rat model when exposed to predominantly progesterone, which resulted in a 2.8-fold attenuation of gene expression induction of cytochrome P450 1A1 (CYP1A1) by TCDD. TCDD and DELAQ, but not laquinimod, concentration-dependently induced CYP1A1 gene expression in both human and rat endometrial models. Interestingly, the relative degree of DELAQ to induce CYP1A1 was higher than that of TCDD in the human model, while it was lower in the rat model. These data clearly show species-differences in response to hormones and AHR ligands between human and rat endometrial cells in vitro, which might greatly affect the applicability of the rat as translational model for human endometrial effects. This warrants further development of human relevant, endometrium-specific test methods for risk assessment purposes.