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Archives of Toxicology (v.87, #4)

Inflammatory findings on species extrapolations: humans are definitely no 70-kg mice by Marcel Leist; Thomas Hartung (pp. 563-567).

Modern toxicology has embraced in vitro methods, and major hopes are based on the Omics technologies and systems biology approaches they bring along (Hartung and McBride in ALTEX 28(2):83–93, 2011; Hartung et al. in ALTEX 29(2):119–28, 2012). A culture of stringent validation has been developed for such approaches (Leist et al. in ALTEX 27(4):309–317, 2010; ALTEX 29(4):373–88, 2012a; Toxicol Res 1:8–22, 2012b), while the quality and usefulness of animal experiments have been little scrutinized. A new study (Seok et al. 2013) now shows the low predictivity of animal responses in the field of inflammation. These findings corroborate earlier findings from comparisons in the fields of neurodegeneration, stroke and sepsis. The low predictivity of animal experiments in research areas allowing direct comparisons of mouse versus human data puts strong doubt on the usefulness of animal data as key technology to predict human safety.

Nanosilver: application and novel aspects of toxicology by Jan K. Schluesener; Hermann J. Schluesener (pp. 569-576).

Nanomaterials are a challenge to toxicology. The high diversity of novel materials and products will require extensive expertize for evaluation and regulatory efforts. Nanomaterials are of substantial scientific and economic potential. Here, we will focus on nanosilver, a material not only with medical applications, but a rapidly increasing use in surprisingly many products. Consequently, toxicological evaluation has to cover an increasing range of complex topics. The toxicology of nanosilver is advancing rapidly; regulatory efforts by Federal Drug Agency and European Environment Protection Agencies are substantial. Current toxicological data, ranging from in vitro studies with cell lines to rodent experiments and ecological evaluation, are numerous, and many groups are providing continuously new data. However, standard classification based on nanosize only is neglecting nanoshape, which adds another level of complexity to the analysis of biological effects. A surprising neglect in nanosilver toxicology so far is the analysis of effects of nanosilver on amyloidosis. Amyloid diseases are widespread in humans and a severe health hazard. The known potential of silver to stimulate amyloidosis in rodents will require a timely and balanced evaluation of nanosilvers.

Keywords: Amyloid; Alzheimer’s disease; Nanotoxicology; Nanoshape; Nanotopography

Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies by J. Fraczek; J. Bolleyn; T. Vanhaecke; V. Rogiers; M. Vinken (pp. 577-610).

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Keywords: Drug safety testing; In vitro models; Primary hepatocyte cultures; Dedifferentiation; Epigenetics; Histone deacetylases; DNA methyltransferases; Liver-enriched transcription factors; MicroRNA

Mechanisms of RAS/β-catenin interactions by Eva Zeller; Katharina Hammer; Melissa Kirschnick; Albert Braeuning (pp. 611-632).

Signaling through the WNT/β-catenin and the RAS (rat sarcoma)/MAPK (mitogen-activated protein kinase) pathways plays a key role in the regulation of various physiological cellular processes including proliferation, differentiation, and cell death. Aberrant mutational activation of these signaling pathways is closely linked to the development of cancer in many organs, in humans as well as in laboratory animals. Over the past years, more and more evidence for a close linkage of the two oncogenic signaling cascades has accumulated. Using different experimental approaches, model systems, and experimental conditions, a variety of molecular mechanisms have been identified by which signal transduction through WNT/β-catenin and RAS interact, either in a synergistic or an antagonistic manner. Mechanisms of interaction comprise an upstream crosstalk at the level of pathway-activating ligands and their receptors, interrelations of cytosolic kinases involved in either pathways, as well as interaction in the nucleus related to the joint regulation of target gene transcription. Here, we present a comprehensive review of the current knowledge on the interaction of RAS/MAPK- and WNT/β-catenin-driven signal transduction in mammalian cells.

Keywords: WNT signaling; MAPK signaling; Crosstalk; Oncogene; ERK; Catenin

The metallohormone cadmium modulates AhR-associated gene expression in the small intestine of rats similar to ethinyl-estradiol by Felix M. Kluxen; Patrick Diel; Nicola Höfer; Eugenia Becker; Gisela H. Degen (pp. 633-643).

Cadmium (Cd) affects the expression of estrogen receptor (ER) and aryl hydrocarbon receptor (AhR)-associated genes in rat uterus and elicits estrogen-like activity in vitro. The small intestine is highly exposed to dietary Cd which may mimic or antagonize estrogen action in this tissue. We investigated the effects of Cd and 17-alpha-ethinylestradiol (EE₂) on AhR-associated gene expression after oral exposure of ovariectomized female Wistar rats, and metallothionein (Mt1a) expression as a typical metal-response marker. Mt1a in the small intestine was strongly induced by co-treatment with CdCl₂ at 2 mg/kg b.wt (Cd 2) and 0.1 mg/kg b.wt EE₂ than by the single compound (3-day gavage). The Cd 2-induced down-regulation of Cyp1a1, Gsta2, and Nqo1 mRNA was not antagonized by pure anti-estrogen (2.5 mg/kg b.wt ZK191703 s.c., ZK). Interestingly, the EE₂-induced down-regulation of Cyp1a1, Gsta2, and Nqo1 mRNA was antagonized by Cd 2 in vivo and in colon cancer cell lines (HT-29 and CaCo-2, treated 5 days with Cd 1 µM and/or E₂ 0.01 µM) with low or no ER-beta expression. Dose dependency was studied after Cd exposure with drinking water (5 and 50 ppm CdCl₂ equivalent to 0.4 and 4 mg/kg b.wt; Cd 0.4, Cd 4) for 28 days and EE₂ as reference. Intestinal Mt1a expression was dose dependently induced, while AhR target genes were down-regulated by Cd 0.4 similar to EE₂ and more pronounced than by Cd 4. We propose that Cd modulates intestinal AhR-associated gene expression similar to estrogens, but (contrary to its effects in uterus) via ER-independent and/or ER-beta-mediated mechanisms. Our new data suggest interference of Cd with estrogen and AhR signaling in the small intestine.

Keywords: Aryl hydrocarbon receptor; Cadmium; Endocrine modulation; Endocrine disruption; Estrogens

Occupational exposure of air crews to tricresyl phosphate isomers and organophosphate flame retardants after fume events by Birgit Karin Schindler; Tobias Weiss; Andre Schütze; Stephan Koslitz; Horst Christoph Broding; Jürgen Bünger; Thomas Brüning (pp. 645-648).

Aircraft cabin air can possibly be contaminated by tricresyl phosphates (TCP) from jet engine oils during fume events. o-TCP, a known neurotoxin, has been addressed to be an agent that might cause the symptoms reported by cabin crews after fume events. A total of 332 urine samples of pilots and cabin crew members in common passenger airplanes, who reported fume/odour during their last flight, were analysed for three isomers of tricresyl phosphate metabolites as well as dialkyl and diaryl phosphate metabolites of four flame retardants. None of the samples contained o-TCP metabolites above the limit of detection (LOD 0.5 μg/l). Only one sample contained metabolites of m- and p-tricresyl phosphates with levels near the LOD. Median metabolite levels of tributyl phosphate (TBP), tris-(2-chloroethyl) phosphate (TCEP) and triphenyl phosphate (TPP) (DBP 0.28 μg/l; BCEP 0.33 μg/l; DPP 1.1 μg/l) were found to be significantly higher than in unexposed persons from the general population. Median tris-(2-chloropropyl) phosphate (TCPP) metabolite levels were significantly not higher in air crews than in controls. Health complaints reported by air crews can hardly be addressed to o-TCP exposure in cabin air. Elevated metabolite levels for TBP, TCEP and TPP in air crews might occur due to traces of hydraulic fluid in cabin air (TBP, TPP) or due to release of commonly used flame retardants from the highly flame protected environment in the airplane. A slight occupational exposure of air crews to organophosphates was shown.

Keywords: Human biomonitoring; Organophosphate flame retardants; TCP; Tricresyl phosphate

Relative oral bioavailability of 3-MCPD from 3-MCPD fatty acid esters in rats by Klaus Abraham; Klaus E. Appel; Edith Berger-Preiss; Elisabeth Apel; Susanne Gerling; Hans Mielke; Otto Creutzenberg; Alfonso Lampen (pp. 649-659).

In order to quantify the relative oral bioavailability of 3-chloropropane-1,2-diol (3-MCPD) from 3-MCPD fatty acid diesters in vivo, 1,2-dipalmitoyl-3-chloropropane-1,2-diol (3-MCPD diester) and 3-MCPD were orally applied to rats in equimolar doses. In both cases, the time courses of 3-MCPD concentrations were measured in blood, various organs, tissues and intestinal luminal contents. The results show that 3-MCPD is released by enzymatic hydrolysis from the 3-MCPD diester in the gastrointestinal tract and distributed to blood, organs and tissues. Based on the measurements in blood, the areas under the curve (AUC) for 3-MCPD were calculated. By comparing both AUC, the relative amount of 3-MCPD bioavailable from the 3-MCPD diester was calculated to be 86 % on average of the amount bioavailable following administration of 3-MCPD. In view of limited experimental data, it is justified for the purpose of risk assessment to assume complete hydrolysis of the diesters in the gastro-intestinal tract. Therefore, assessment of the extent of exposure to 3-MCPD released from its fatty acid esters should be performed in the same way as exposure to the same molar quantity of 3-MCPD.

Keywords: 3-MCPD fatty acid ester; Risk assessment; Toxicokinetics; Bioavailability; Enzymatic hydrolysis

A physiologically based pharmacokinetic model for the oxime TMB-4: simulation of rodent and human data by Teresa R. Sterner; Christopher D. Ruark; Tammie R. Covington; Kyung O. Yu; Jeffery M. Gearhart (pp. 661-680).

Multiple oximes have been synthesized and evaluated for use as countermeasures against chemical warfare nerve agents. The current U.S. military and civilian oxime countermeasure, 2-[(hydroxyimino)methyl]-1-methylpyridin-1-ium chloride (2-PAM), is under consideration for replacement with a more effective acetylcholinesterase reactivator, 1,1’-methylenebis{4-hydroxyiminomethyl}pyridinium dimethanesulfonate (MMB-4). Kinetic data in the scientific literature for MMB-4 are limited; therefore, a physiologically based pharmacokinetic (PBPK) model was developed for a structurally related oxime, 1,1’-trimethylenebis{4-hydroximinomethyl}pyridinium dibromide. Based on a previous model structure for the organophosphate diisopropylfluorophosphate, the model includes key sites of acetylcholinesterase inhibition (brain and diaphragm), as well as fat, kidney, liver, rapidly perfused tissues and slowly perfused tissues. All tissue compartments are diffusion limited. Model parameters were collected from the literature, predicted using quantitative structure–property relationships or, when necessary, fit to available pharmacokinetic data from the literature. The model was parameterized using rat plasma, tissue and urine time course data from intramuscular administration, as well as human blood and urine data from intravenous and intramuscular administration; sensitivity analyses were performed. The PBPK model successfully simulates rat and human data sets and has been evaluated by predicting intravenous mouse and intramuscular human data not used in the development of the model. Monte Carlo analyses were performed to quantify human population kinetic variability in the human evaluation data set. The model identifies potential pharmacokinetic differences between rodents and humans, indicated by differences in model parameters between species. The PBPK model can be used to optimize the dosing regimen to improve oxime therapeutic efficacy in a human population.

Keywords: Oxime; PBPK model; Rat; Human; Pharmacokinetic; Organophosphate

AhR-mediated changes in global gene expression in rat liver progenitor cells by Dagmar Faust; Jan Vondráček; Pavel Krčmář; Lenka Šmerdová; Jiřina Procházková; Eva Hrubá; Petra Hulinková; Bernd Kaina; Cornelia Dietrich; Miroslav Machala (pp. 681-698).

Although the tumor-promoting effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar polychlorinated biphenyls (PCBs), and related compounds in liver tissue are primarily attributed to the activation of the aryl hydrocarbon receptor (AhR), the underlying molecular mechanisms are still unclear. Liver progenitor (oval) cells have been suggested to constitute a potential target for hepatocarcinogenic chemicals. To better understand AhR-driven pathways, we analyzed the transcriptional program in response to coplanar PCB 126 in contact-inhibited rat liver progenitor WB-F344 cells using high-density microarrays. After 6-h treatment, we identified 145 significantly deregulated genes considered to be direct AhR-dependent target genes. The number of differentially regulated genes increased to 658 and 968 genes after 24 and 72 h, respectively. Gene ontology analysis revealed that these genes were primarily involved in drug and lipid metabolism, cell cycle and growth control, cancer developmental processes, cell–cell communication, and adhesion. Interestingly, the Wnt and TGF-β signaling pathways, both being involved in developmental and tumorigenic processes, belonged to the most affected pathways. AhR- and ARNT-dependent regulation of selected target genes of interest was then confirmed using TCDD as a model AhR agonist, together with pharmacological inhibition of the AhR and by RNA-interference techniques. We demonstrated AhR-dependent regulation of emerging and novel AhR target genes, such as Fst, Areg, Hbegf, Ctgf, Btg2, and Foxq1. Among them, the transcription factor Foxq1, recently suggested to contribute to tumor promotion and/or progression, was found to be regulated at both mRNA and protein levels by AhR/ARNT activation.

Keywords: Aryl hydrocarbon receptor; TCDD; PCB 126; Gene expression; Rat liver progenitor cells

Excision repair of BPDE-adducts in human lymphocytes: diminished capacity associated with ERCC1 C8092A (rs3212986) polymorphism by Tao Yu; Yanhua Liu; Xiaobo Lu; Sha Xiao; Yuan Cai; Cuihong Jin; Qiufang Liu; Jinghua Yang; Shengwen Wu; Xiaoyan Bao; Liang Pan; Tahar van der Straaten (pp. 699-709).

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a metabolite of Benzo[a]pyrene (B[a]P), is a high-risk factor for development of a number of cancers. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair system of which ERCC1 exerts an important role. We investigated whether two single nucleotide polymorphisms in ERCC1 (C19007T; rs11615 and C8092A; rs3213986) affected the repair efficacy of BPDE-DNA adducts. We collected peripheral blood of 780 healthy individuals from the northeast of China and detected the genotypes of rs11615 and rs3213986. The amount of induced BPDE-DNA adducts in lymphocytes from 117 randomly selected participants was assessed by HPLC. Presence of BPDE-DNA adducts in nucleus of lymphocytes was visualized using the modified comet assay. ERCC1 and CAST (3′ adjacent gene of ERCC1) mRNA expression levels were quantified after in vitro exposure to BPDE. We found that the minor A allele in rs3212986 was related to higher levels of BPDE-DNA adducts and holistic marking DNA damage (P < 0.01). Haplotype CA (rs11615 and rs3213986) was also associated with an elevated risk of high BPDE-DNA adduct levels (OR = 1.801, 95 % CI of OR 1.191–2.724). Interestingly, in participants with AA genotype for rs3213986, CAST mRNA level was decreased compared to individuals with the homozygous CC genotype. Our findings suggests that ERCC1 C8092A (rs3213986) is associated with a diminished capacity of repairing BPDE-DNA adducts and may be used as a valid biomarker to predict an individual’s risk to develop cancer upon exposure to environmental carcinogens.

Keywords: ERCC1; CAST; Single nucleotide polymorphism; BPDE-DNA adduct; DNA repair capacity

The summary on non-reactivation cholinergic properties of oxime reactivators: the interaction with muscarinic and nicotinic receptors by O. Soukup; D. Jun; G. Tobin; K. Kuca (pp. 711-719).

Organophosphorus inhibitors (OP) of acetylcholinesterase (AChE) represent a group of highly toxic compounds. The treatment of OP intoxication is, however, insufficiently ensured. Currently, two main categories of drugs—anticholinergics and oxime reactivators— are employed as antidotes. Oximes have been reported to act at several levels of the cholinergic transmission, and among the non-reactivation effects, the interaction with cholinergic receptors stands out. This review addresses issues correlated with non-reactivating effects of oxime reactivators with a special focus on the muscarinic and nicotinic receptors, but involvement of other cholinergic structures such as AChE and choline uptake carriers are discussed too. It can be concluded that the oxime reactivators show a variation in their antagonistic effect on the muscarinic and nicotinic receptors, which is likely to be of significance in the treatment of OP poisoning. In vitro data reported oximes to exert higher efficacy on the muscarinic M2 subtype than on the AChE. However, this effect seemed to be subtype specific since the antagonistic M3 effect was lower. Also, and importantly, the antimuscarinic effect was larger than that on nicotinic receptors. Even though atropine showed a much higher muscarinic antagonism, it is supposed that non-reactivation properties of oxime reactivators play a significant role in the treatment of OP poisoning.

Keywords: Oxime reactivators; Organophosphate; Non-reactivation; Muscarinic receptor; Nicotinic receptor; Anticholinergics

A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles by Lisa Hoelting; Benjamin Scheinhardt; Olesja Bondarenko; Stefan Schildknecht; Marion Kapitza; Vivek Tanavde; Betty Tan; Qian Yi Lee; Stefan Mecking; Marcel Leist; Suzanne Kadereit (pp. 721-733).

Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood–brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6⁺ precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

Keywords: Human embryonic stem cells; Neurospheres; Developmental neurotoxicity (DNT); Polyethylene nanoparticles; Methylmercury

Endocrine disruptors and bone metabolism by Dimitrios Agas; Maria Giovanna Sabbieti; Luigi Marchetti (pp. 735-751).

Bone microenvironment is a complex dynamic equilibrium between osteoclasts and osteoblasts and is modulated by a wide variety of hormones and osteocyte mediators secreted in response to physiological and pathological conditions. The rate of remodeling involves tight coupling and regulation of both cells population and is regulated by a wide variety of hormones and mediators such as parathyroid hormone, prostaglandins, thyroid hormone, sex steroids, etc. It is also well documented that bone formation is easily influenced by the exposure of osteoblasts and osteoclasts to chemical compounds. Currently, humans and wildlife animals are exposed to various environmental xenoestrogens typically at low doses. These compounds, known as endocrine disruptor chemicals (EDCs), can alter the systemic hormonal regulation of the bone remodeling process and the skeletal formation. This review highlights the effects of the EDCs on mammalian bone turnover and development providing a macro and molecular view of their action.

Keywords: Endocrine disruptors; Skeletal formation; Bone remodeling; Osteoblasts; Osteoclasts

Impairment of novel object recognition in adulthood after neonatal exposure to diazinon by Tin-Tin Win-Shwe; Daisuke Nakajima; Sohel Ahmed; Hidekazu Fujimaki (pp. 753-762).

Diazinon is an organophosphate pesticide that is still heavily used in agriculture, home gardening, and indoor pest control in Japan. The present study investigated the effect of neonatal exposure to diazinon on hippocampus-dependent novel object recognition test performance and the expression of the N-methyl-d-aspartate (NMDA) receptor and its signal transduction pathway-related genes in the hippocampi of young adult and adult mice. Male offspring of C3H/HeN mice were subcutaneously treated with 0, 0.5, or 5 mg/kg of diazinon for 4 consecutive days beginning on postnatal day (PND) 8. Beginning on PND 46 or PND 81, a novel object recognition test was performed on 4 consecutive days. The hippocampi were collected on PND 50 or PND 85 after the completion of the novel object recognition test, and the expression levels of neurotrophins and the NMDA receptor and its signal transduction pathway-related genes were examined using real-time RT-PCR. Diazinon-injected mice exhibited a poor ability to discriminate between novel and familiar objects during both the PND 49 and the PND 84 tests. The NMDA receptor subunits NR1 and NR2B and the related protein kinase calcium/calmodulin-dependent protein kinase (CaMK)-IV and the transcription factor cyclic AMP responsive element binding protein (CREB)-1 mRNA levels were reduced in the PND 50 mice. However, no significant changes in the expressions of the NMDA subunits and their signal transduction molecules were observed in the hippocampi of the PND 85 mice. The expression level of nerve growth factor mRNA was significantly reduced in the PND 50 or 85 mice. These results indicate that neonatal diazinon exposure impaired the hippocampus-dependent novel object recognition ability, accompanied by a modulation in the expressions of the NMDA receptor and neurotrophin in young adult and adult mice.

Keywords: Neonate; Diazinon; Hippocampus; Object recognition; Mice

Induction of skin sensitization is augmented in Nrf2-deficient mice by Jochem W. van der Veen; Eric R. Gremmer; Jolanda P. Vermeulen; Henk van Loveren; Janine Ezendam (pp. 763-766).

Several in vitro DNA microarray studies have shown the importance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in skin sensitization. Nevertheless, the exact in vivo role of the Nrf2-Keap1 pathway during the induction of skin sensitization remains unknown. To study the function of Nrf2, a local lymph node assay was performed in wild-type and Nrf2-deficient mice using 2,4-dinitrochlorobenzene. The Nrf2-deficient mice show a more pronounced response, indicating that Nrf2 is involved in dampening the induction of skin sensitization.

Keywords: Skin sensitization; LLNA; Nrf2-deficient; In vivo

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Archives of Toxicology (v.87, #4)