Mixture effects at very low doses with combinations of anti-androgenic pesticides, antioxidants, industrial pollutant and chemicals used in personal care products

This article has been made available through the Brunel Open Access Publishing Fund.Many xenobiotics have been identified as in vitro androgen receptor (AR) antagonists, but information about their ability to produce combined effects at low concentrations ismissing. Such data can reveal whether joint effects at the receptor are induced at low levels andmay support the prioritisation of in vivo evaluations and provide orientations for the grouping of anti-androgens in cumulative risk assessment. Combinations of 30 AR antagonists from a wide range of sources and exposure routes (pesticides, antioxidants, parabens, UV-filters, synthetic musks, bisphenol-A, benzo(a)pyrene, perfluorooctane sulfonate and pentabromodiphenyl ether) were tested using a reporter gene assay (MDA-kb2). Chemicalswere combined at threemixture ratios, equivalent to single components' effect concentrations that inhibit the action of dihydrotesterone by 1%, 10% or 20%. Concentration addition (CA) and independent action were used to calculate additivity expectations. We observed complete suppression of dihydrotestosterone effects when chemicals were combined at individual concentrations eliciting 1%, 10% or 20% AR antagonistic effect. Due to the large number of mixture components, the combined AR antagonistic effects occurred at very low concentrations of individual mixture components. CA slightly underestimated the combined effects at all mixture ratios. In conclusion, large numbers of AR antagonists froma wide variety of sources and exposure routes have the ability of acting together at the receptor to produce joint effects at very low concentrations. Significant mixture effects are observed when chemicals are combined at concentrations that individually do not induce observable AR antagonistic effects. Cumulative risk assessment for AR antagonists should apply grouping criteria based on effects where data are available, rather than on criteria of chemical similarity

Mind the gap: Can we explain declining male reproductive health with known antiandrogens?

This article has been made available through the Brunel Open Access Publishing Fund.Several countries have experienced rises in cryptorchidisms, hypospadias and testicular germ cell cancer. The reasons for these trends are largely unknown, but Skakkebaek has proposed that these disorders form a testicular dysgenesis syndrome and can be traced to androgen insufficiency in foetal life. This suggests that antiandrogenic chemicals might contribute to risks, but few chemicals have been linked to these diseases in epidemiological studies. In animal studies with p,p0-dichlorodiphenyldichloroethylene, effects typical of disruptions of male sexual differentiation became apparent when the foetal levels of this androgen receptor (AR) antagonist approached values associated with responses in in vitro assays. This prompted us to analyse whether the 22 chemicals with AR antagonistic properties would produce mixture effects in an in vitro AR antagonism assay when combined at concentrations found in human serum. Other antiandrogenic modalities could not be considered. Two scenarios were investigated, one representative of average serum levels reported in European countries, the other in line with levels towards the high exposures. In both situations, the in vitro potency of the 22 selected AR antagonists was too low to produce combined AR antagonistic effects at the concentrations found in human serum, although the high exposure scenario came quite close to measurable effects. Nevertheless, our analysis exposes an explanation gap which can only be bridged by conjuring up as yet undiscovered high potency AR antagonists or, alternatively, high exposures to unknown agents of average potency

A Graph Transformational View on Reductions in NP

Many decision problems in the famous and challenging complexity class NP are graph problems and can be adequately specified by polynomial graph transformation units. In this paper, we propose to model the reductions in NP by means of a special type of polynomial graph transformation units, too. Moreover, we present some first ideas how the semantic requirements of reductions including their correctness can be proved in a systematic way

Purification and structural characterization of the Na⁺-translocating ferredoxin: NAD⁺ reductase (Rnf) complex of Clostridium tetanomorphum

Various microbial metabolisms use H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD+ for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na+ translocation is provided by orchestrating Na+ uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

This article is made available through the Brunel Open Access Publishing Fund. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration–response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.United Kingdom Food Standards Agenc

Rydberg-London Potential for Diatomic Molecules and Unbonded Atom Pairs

We propose and test a pair potential that is accurate at all relevant distances and simple enough for use in large-scale computer simulations. A combination of the Rydberg potential from spectroscopy and the London inverse-sixth-power energy, the proposed form fits spectroscopically determined potentials better than the Morse, Varnshi, and Hulburt-Hirschfelder potentials and much better than the Lennard-Jones and harmonic potentials. At long distances, it goes smoothly to the correct London force appropriate for gases and preserves van der Waals's "continuity of the gas and liquid states," which is routinely violated by coefficients assigned to the Lennard-Jones 6-12 form.Comment: Five pages, 10 figure

Declining semen quality and polybrominated diphenyl ethers (PBDEs): Review of the literature to support the derivation of a reference dose for a mixture risk assessment

Appendix A. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijheh.2022.113953.Copyright © 2022 The Authors. To support a mixture risk assessment for chemicals that interfere with male reproductive health, we reviewed the literature to identify studies of polybrominated diphenyl ethers (PBDEs) and poor semen quality. Several epidemiological studies have shown associations of PBDE exposures with declining semen quality, non-descending testes and penile malformations. In rodent studies, poor semen quality, changes in testosterone levels and reproductive tissues have been observed. In vitro studies with reporter gene constructs show PBDE congeners as androgen receptor antagonists, and mixture studies in these systems have demonstrated that PBDE congeners act together with other androgen receptor antagonists. These observations led us to attempt the estimation of reference doses for specific PBDE congeners that can be used in a future mixture risk assessment for deteriorations of semen quality. While epidemiological studies provide support for such associations, they were uninformative for derivations of reference doses, due to the incompatibility of dose metrics used in exposure assessments. We therefore based our estimates on animal studies. Using a rigorous confidence rating approach, we found robust evidence that BDE-47 produced reductions in semen quality. We identified only one high confidence study of BDE-99 and accordingly evaluated the strength of evidence as moderate. One high confidence, and several medium confidence experimental studies observed declines in semen quality after BDE-209 exposure. Using established risk assessment procedures, we estimated that BDE-47 exposures below 0.15 μg/kg/d are unlikely to lead to reductions in semen quality. The corresponding exposures for BDE-99 and BDE-209 are 0.003 μg/kg/d and 1000 μg/kg/d. It is planned to use these estimates as reference doses in a mixture risk assessment of deteriorations in semen quality, involving multiple other chemicals also contributing to poor semen quality.This work was conducted with funding from the European HBM4EU project (www.hbm4eu.eu), contract number 733032, Horizon 2020 programme, which is gratefully acknowledged

Genotoxic mixtures and dissimilar action: Concepts for prediction and assessment

This article has been made available through the Brunel Open Access Publishing Fund. This article is distributed under the terms of the creative commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s)and the source are credited.Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.UK Food Standards Agenc

Recent Advances in 3D printing for in vitro cancer models

This paper is part of the special topic, Physics of 3D Printing.3D printing techniques allow for the precise placement of living cells, biological substances, and biochemical components, establishing themselves as a promising approach in bioengineering. Recently, 3D printing has been applied to develop human-relevant in vitro cancer models with highly controlled complexity and as a potential method for drug screening and disease modeling. Compared to 2D culture, 3D-printed in vitro cancer models more closely replicate the in vivo microenvironment. Additionally, they offer a reduction in the complexity and ethical issues associated with using in vivo animal models. This focused review discusses the relevance of 3D printing technologies and the applied cells and materials used in cutting-edge in vitro cancer models and microfluidic device systems. Future prospective solutions were discussed to establish 3D-printed in vitro models as reliable tools for drug screening and understanding cancer disease mechanisms.Brunel Research Interdisciplinary Labs (BRIL) and Brief Award (BRIEF