Thresholds of adversity for endocrine disrupting substances: a conceptual case study.

For endocrine disrupting chemicals (EDC) the existence of "safe exposure levels", that is exposure levels that do not present an appreciable risk to human health is most controversially discussed, as is the existence of health-based reference values. Concerns have been especially raised that EDCs might not possess a threshold level such that no exposure level to EDCs can be considered safe. To explore whether or not threshold levels can be identified, we performed a screening exercise on 14 pesticidal and biocidal active substances previously identified as EDCs in the European Union. The respective substances are ideal subjects for case studies to review for endocrine activity and disruptive potential following well-defined regulatory assessment based on solid data to effectually establish adversity as consequence of endocrine disruption. Dimethomorph, metiram and propiconazole for which the weight of evidence demonstrating endocrine disruption was the strongest were used as subjects for further study. Epoxiconazole was additionally selected as its effects on the endocrine system are extensive. For all four substances, analysis of the toxicological data clearly indicated thresholds of adversity below which no adverse effects mediated through an endocrine mechanism were observed. Particular emphasis was placed on mechanistic considerations including homeostasis and the concept of adversity. As a proof of concept this study provides evidence that like other substances of toxicological concern EDCs have threshold levels for adversity. While for some EDCs the respective thresholds might indeed be very low this shows that, data allowing, for other EDCs sufficiently protective reference values can be derived.

Pathway and time-resolved benzo[a]pyrene toxicity on Hepa1c1c7 cells at toxic and subtoxic exposure.

Benzo[a]pyrene (B[a]P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 µM) or a subtoxic (50 nM) concentration of B[a]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B[a]P, which was followed by quantitative analysis of the concentration of intracellular B[a]P and seven representative degradation products. Exposure with 5 µM B[a]P led to a maximal intracellular concentration of 1604 pmol/5 × 10(4) cells, leveling at 55 pmol/5 × 10(4) cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B[a]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 µM), and 150 (only 50 nM) proteins were found to be regulated in response to B[a]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B[a]P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B[a]P specific effects.

Transcriptional signatures of regulatory and toxic responses to benzo-[a]-pyrene exposure.

BACKGROUND: Small molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses ("side effects"). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals. RESULTS: We explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and biological function. Finally, the accuracy of the predictions was supported through extensive experimental validation. CONCLUSION: Using a combination of machine learning followed by extensive experimental validation, we have further expanded the known catalog of genes regulated by the environmentally sensitive transcription factor Ahr. More broadly, this study presents a strategy for distinguishing receptor-dependent responses and side effects based on expression time courses.

Toxicity profiles of four metals and 17 xenobiotics in the human hepatoma cell line HepG2 and the protozoa Tetrahymena pyriformis--a comparison.

We performed an interspecies comparison for the human hepatoma cell line HepG2 and the eukaryotic single cell organism Tetrahymena pyriformis (T. pyriformis) for 17 xenobiotics with diverse structures and four metals. The cytotoxicity was assessed by four different cell viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction (MTT), neutral red uptake (NRU), resazurin dye (AlamarBlue), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)) for the HepG2 and by cell count and MTT for T. pyriformis. For HepG2 cells, the results revealed interassay variations depending on the compound. The highest assay conformity was found for the metal Hg(2+) and the fungicide prochloraz. The AlamarBlue assay was the most sensitive assay according to low-effect concentrations. By contrast, the NRU assay was comprised of more frequent whole concentration response relationships and was more susceptible to EC(50). For T. pyriformis the EC(50) values of the two applied assays displayed a high conformity (R(2) = 0.97). Comparing the EC(50) values obtained by the MTT assay for the two cell models, a direct correlation was absent for the xenobiotics and only present for the metals (Cd(2+), Cu(2+), and Ni(2+)). Moreover, the protozoa T. pyriformis displayed a 20 times higher sensitivity than the cell line. The highest interspecies difference of three log degrees was obtained for the polycyclic aromatic hydrocarbon fluoranthene. In addition, a correlation of the EC(50) values and octanol-water partition coefficient (log K(OW)) of the xenobiotics was performed. No correlation was found for HepG2, and a weak one for T. pyriformis. Interestingly, the interspecies difference of logarithmized EC(50) correlated positive with the log K(OW) (R(2) = 0.65). In conclusion, to obtain reliable evidence for human cytotoxicity, more than one viability/cytotoxicity assay had to be applied for cell lines. Second, the human hepatoma cell line was less affected by the organic compounds than the eukaryotic single-cell organism and was also less dependent on the log K(OW) of the xenobiotic.

Diversity of common alternative splicing variants of human cytochrome P450 1A1 and their association to carcinogenesis.

Cytochrome P450 1A1 (CYP1A1) belongs to the enzymes of biotransformation of phase I. CYP1A1 performs the catalytic activation of exogenous and endogenous substrates to more carcinogenic metabolites. Overexpression of the wild-type and a recently described splice variant (CYP1A1v, ovarian cancer) are attributed to neoplastic transformation. Here we describe novel CYP1A1 splicing variants commonly and frequently transcribed in leucocytes of healthy volunteers, separated from variants exclusively expressed in tumour cell lines. Interestingly, all the novel splicing variants in leukocytes are generated by employing of two nested splice site pairs, one outer canonical and one inner non-canonical splice site pair, within the exon 2 of the human CYP1A1. In general, the frequent presence of common splicing variants in healthy volunteers has to be consider as a physiological feature of human CYP1A1 transcription process, rather than a signature of carcinogenesis.

Influence of nanoparticle-mediated transfection on proliferation of primary immune cells in vitro and in vivo.

INTRODUCTION: One of the main obstacles in the widespread application of gene therapeutic approaches is the necessity for efficient and safe transfection methods. For the introduction of small oligonucleotide gene therapeutics into a target cell, nanoparticle-based methods have been shown to be highly effective and safe. While immune cells are a most interesting target for gene therapy, transfection might influence basic immune functions such as cytokine expression and proliferation, and thus positively or negatively affect therapeutic intervention. Therefore, we investigated the effects of nanoparticle-mediated transfection such as polyethylenimine (PEI) or magnetic beads on immune cell proliferation. METHODS: Human adherent and non-adherent PBMCs were transfected by various methods (e.g. PEI, Lipofectamine® 2000, magnetofection) and stimulated. Proliferation was measured by lymphocyte transformation test (LTT). Cell cycle stages as well as expression of proliferation relevant genes were analyzed. Additionally, the impact of nanoparticles was investigated in vivo in a murine model of the severe systemic immune disease GvHD (graft versus host disease). RESULTS: The proliferation of primary immune cells was influenced by nanoparticle-mediated transfection. In particular in the case of magnetic beads, proliferation inhibition coincided with short-term cell cycle arrest and reduced expression of genes relevant for immune cell proliferation. Notably, proliferation inhibition translated into beneficial effects in a murine GvHD model with animals treated with PEI-nanoparticles showing increased survival (pPEI = 0.002) most likely due to reduced inflammation. CONCLUSION: This study shows for the first time that nanoparticles utilized for gene therapeutic transfection are able to alter proliferation of immune cells and that this effect depends on the type of nanoparticle. For magnetic beads, this was accompanied by temporary cell cycle arrest. Notably, in GvHD this nonspecific anti-proliferative effect might contribute to reduced inflammation and increased survival.

The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2.

Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2. The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide. The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively. In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.