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Archives of Toxicology (v.85, #2)

Stem cells in toxicity testing by J. D. Stewart; H. M. Bolt (pp. 77-78).

Present state and future perspectives of using pluripotent stem cells in toxicology research by Anna M. Wobus; Peter Löser (pp. 79-117).

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed.

Keywords: Embryonic stem (ES) cells; Embryonic carcinoma (EC) cells; Induced pluripotent stem (iPS) cells; Mouse; Human; Pluripotency; Differentiation; Embryonic stem cell test (EST); In vitro embryotoxicity; Cardiotoxicity; Hepatotoxicity

Genetic variants associated with arsenic metabolism within human arsenic (+3 oxidation state) methyltransferase show wide variation across multiple populations by Junko Fujihara; Toshihiro Yasuda; Hideaki Kato; Isao Yuasa; Arturo Panduro; Takashi Kunito; Haruo Takeshita (pp. 119-125).

Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. The objective of this study was to investigate the diversity of the AS3MT gene in Mexican and German populations. The distribution of 18 single nucleotide polymorphisms (SNPs) in AS3MT was assessed on healthy individuals: 38 Mestizo, 69 Nahuas, 50 Huicholes, and 32 Germans. All 18 SNPs were polymorphic in the German and Mexican populations. Of the three Mexican populations, a minor allele frequency was the highest in the Mestizo, followed by the Nahuas and Huicholes. In the German and three Mexican groups, haplotype #1(TATAGAAGTCTTCATGAC) was the most predominant. Seven haplotypes were newly found in the German and three Mexican populations. The D′ values between SNP pairs were high in the German and Nahua populations; they had a similar pattern. The pattern of the Mestizo was more similar to the African than to the other Mexican populations. Huicholes had a moderate pattern of the African and German/Nahua populations. The network had three clusters. One originated in the African population and another may have originated in an Asian (Chinese and/or Japanese) population. The third one may have originated among Caucasians. This study is the first to demonstrate the existence of genetic heterogeneity in the distribution of 18 SNPs in AS3MT of German and Mexican populations.

Keywords: Arsenic (+3 oxidation state) methyltransferase (AS3MT); Ethnic differences; Genetic variation; Haplotype; Linkage disequilibrium; Single nucleotide polymorphisms (SNPs); Mexican; German

Determination of 2,5-toluylenediamine (2,5-TDA) and aromatic amines in urine after personal application of hair dyes: kinetics and doses by Thomas Schettgen; K. Heinrich; T. Kraus; Monika Gube (pp. 127-133).

The personal use of hair dye products is currently under discussion due to the potentially increased risk of bladder cancer among long-time users described in epidemiological literature. In order to investigate the dermal absorption of aromatic diamines as well as aromatic amines possibly present as contaminants in hair dye formulations, we conducted a biomonitoring study under real-life conditions and calculated kinetics and doses for the urinary excretion. Urine samples of two female subjects were collected for a time period of 48 h after personal application of a hair dye cream and analysed for aromatic diamines as well as o-toluidine and 4-aminobiphenyl using highly specific GC/MS-methods. 2,5-Toluylenediamine (2,5-TDA) as active ingredient of hair dyes is rapidly absorbed dermally. After a distribution phase of 12 h, 2,5-TDA is excreted with a half-time of 8 h. Excretion was 90% complete within 24 h after application. The doses of 2,5-TDA excreted within 48 h were 700 μg for application of a brown-reddish hair dye cream and 1.5 mg for the application of a brown-black hair dye cream. Urinary 4-aminobiphenyl as well as contaminations with other aromatic diamines were not detectable in our study. Due to the artifactual formation of o-toluidine in the presence of high concentrations of urinary 2,5-TDA, our results could not prove an increased internal exposure of humans to carcinogenic amines after personal application of hair dyes.

Keywords: Hair dye; Aromatic diamines; Aromatic amines; Biological monitoring; o-Toluidine; Urine; Toxicokinetics

Marine brevetoxin induces IgE-independent mast cell activation by Susana C. Hilderbrand; Rachel N. Murrell; James E. Gibson; Jared M. Brown (pp. 135-141).

Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H₁-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow–derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (β-hexosaminidase release), intracellular Ca²⁺ and Na⁺ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²⁺ and Na⁺ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na⁺ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.

Keywords: Brevetoxin; Mast cell; Immune

Induction of DNA strand breaks by dental composite components compared to X-ray exposure in human gingival fibroblasts by Jürgen Durner; Małgorzata Dębiak; Alexander Bürkle; Reinhard Hickel; Franz-Xaver Reichl (pp. 143-148).

The toxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to degradation processes or incomplete polymerization. Some of these eluted substances have a genotoxic potential. We tested the hypothesis that realistic concentrations (and/or worst case concentrations/situations) of bisphenol-A-glycidyldimethacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) found in elution experiments can cause DNA strand breaks in human gingival fibroblasts (HGF). Such DNA damage was compared with that resulting from ionizing radiation coming from natural sources, dental radiography or tumor therapy. TEGDMA, HEMA and MMA did not induce DNA strand breaks at concentrations of up to 10 mM. About 24 h after incubation with 0.25 mM BisGMA, significantly more DNA strand breaks were found in HGF compared to controls. DNA strand breaks caused by 0.25 mM BisGMA, correspond to DNA strand breakage caused by irradiation with 4 Gy, only used in the high single-dose irradiation tumor therapy. But 0.25 mM BisGMA is more than 100-fold higher than that concentration found in worst case calculations. Therefore, our data did not support our hypothesis.

Keywords: TEGDMA; HEMA; DNA-strand breaks; X-ray; Genotoxic potential

Effects of extracellular acidic–alkaline stresses on trigeminal ganglion neurons in the mouse embryo in vivo by Shingo Mukai; Hiroshi Nakagawa; Hiroyuki Ichikawa; Shuichi Miyazaki; Kazuhiko Nishimura; Saburo Matsuo (pp. 149-154).

Acidic–alkaline stresses caused by ischemia and hypoglycemia induce neuronal cell death resulting from intracellular pH disturbance. The effects of acidic–alkaline disturbance on the trigeminal ganglion (TG) neurons of the embryonic mouse were investigated by caspase-3-immunohistochemistry and Nissl staining. TG neurons exhibited apoptosis in 3.08 ± 0.55% of neurons in intact embryos at day 16. Intraperitoneal injection of alkaline solution (pH 8.97; 0.005–0.1 M K₂HPO₄ or 0.01–0.04 M KOH) into the embryo at embryonic day 15 significantly increased the number of apoptotic neurons in the TG at embryonic day 16 with dependence on concentration (3.40–6.05 and 2.93–5.55%, respectively). On the other hand, acidic solutions (pH 4.4; 0.01–0.2 M KH₂PO₄) slightly, but not significantly, increased the number of apoptotic cells (3.64–5.15%, without dependence on concentration). Neutral solutions (pH 7.4; 0.01–0.2 M potassium phosphate buffer) had no effect on neuronal survival in the TG (2.89–3.48%). The results indicated that alkaline stress significantly increased apoptosis in the developing nervous system, but acidic stress did not.

Keywords: Cell death; Excitotoxicity; Extracellular ions; Neurotoxicity; Embryogenesis

Immunohistochemical analyses at the late stage of tumor promotion by oxfendazole in a rat hepatocarcinogenesis model by Yasuaki Dewa; Jihei Nishimura; Meilan Jin; Masaomi Kawai; Yukie Saegusa; Sayaka Kenmochi; Keisuke Shimamoto; Tomoaki Harada; Makoto Shibutani; Kunitoshi Mitsumori (pp. 155-162).

The present study was performed to characterize immunohistochemically the expression levels of molecules related to not only xenobiotic and antioxidant functions but also cell proliferation and apoptosis in neoplastic lesions induced by the benzimidazole anthelmintic, oxfendazole (OX), at the late stage of its tumor promotion in a rat hepatocarcinogenesis model. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and 2 weeks later they were fed a diet containing 0% (basal diet) or 0.05% OX for 26 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and killed at week 28. Histopathologically, OX increased the incidence and multiplicity of altered foci (4.0- and 3.6-fold, respectively) and hepatocellular adenomas (HCAs) (3.0- and 5.5-fold, respectively). OX treatment induced 5.2- and 5.6-fold increases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and single-stranded DNA (ssDNA)-positive cells in HCAs compared with the surrounding tissue, respectively. Staining for the cell cycle regulators P21 and C/EBPα and the AhR-regulated CYP1A1 molecules decreased but increased reactivity of the Nrf2-regulated, detoxifing/antioxidant molecules aldo–keto reductase 7 (AKR7) and glutathione peroxidase 2 (GPX2) were also seen in HCAs compared with the surrounding hepatocytes. These results suggest that dysregulation of cell proliferation and apoptosis and escape from oxidative stress elicited by OX treatment play an important role in OX-induced hepatocarcinogenesis in rats.

Keywords: Oxfendazole; Oxidative stress; Cell proliferation; Apoptosis; CYP1A enzyme induction; Hepatocarcinogenesis

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Archives of Toxicology (v.85, #2)