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Archives of Toxicology (v.86, #5)

Toxicology of magnetic nanoparticles: disturbed body iron homeostasis? by R. Marchan; R. Reif; J. G. Hengstler (pp. 683-684).

Magnetic nanoparticles: an update of application for drug delivery and possible toxic effects by Ji-Eun Kim; Ji-Young Shin; Myung-Haing Cho (pp. 685-700).

Magnetic nanoparticles (MNPs) represent a subclass within the overall category of nanomaterials and are widely used in many applications, particularly in the biomedical sciences such as targeted delivery of drugs or genes, in magnetic resonance imaging, and in hyperthermia (treating tumors with heat). Although the potential benefits of MNPs are considerable, there is a distinct need to identify any potential toxicity associated with these MNPs. The potential of MNPs in drug delivery stems from the intrinsic properties of the magnetic core combined with their drug loading capability and the biomedical properties of MNPs generated by different surface coatings. These surface modifications alter the particokinetics and toxicity of MNPs by changing protein–MNP or cell–MNP interactions. This review contains current advances in MNPs for drug delivery and their possible organ toxicities associated with disturbance in body iron homeostasis. The importance of protein–MNP interactions and various safety considerations relating to MNP exposure are also addressed.

Keywords: Magnetic nanoparticle; Drug delivery; Toxicity; Review

Toxicity studies with 5-hydroxymethylfurfural and its metabolite 5-sulphooxymethylfurfural in wild-type mice and transgenic mice expressing human sulphotransferases 1A1 and 1A2 by Morana Bauer-Marinovic; Felicitas Taugner; Simone Florian; Hansruedi Glatt (pp. 701-711).

5-Sulphooxymethylfurfural (SMF), an electrophilic metabolite of the abundant Maillard product 5-hydroxymethylfurfural (HMF), was intraperitoneally administered to FVB/N mice. At a dosage of 250 mg/kg, most animals died after 5–11 days due to massive damage to proximal tubules. At lower dosages, administered repeatedly, tubules also were the major target of toxicity, with regeneration and atypical hyperplasia occurring at later periods. Additionally, hepatotoxic effects and serositis of peritoneal tissues were observed. SMF is a minor metabolite of HMF in conventional mice, but HMF is an excellent substrate for a major sulphotransferase (hSULT1A1) in humans. Parental FVB/N mice and FVB/N-hSULT1A1/2 mice, carrying multiple copies of the hSULT1A1/2 gene cluster, were exposed to HMF in drinking water (0, 134 and 536 mg/kg body mass/day) for 12 weeks. Nephrotoxic effects and enhanced proliferation of hepatocytes were only detected at the high dosage. They were mild and, surprisingly, unaffected by hSULT1A1/2 expression. Thus, SMF was a potent nephrotoxicant when administered as a bolus, but did not reach levels sufficient to produce serious toxicity when generated from HMF administered continuously via drinking water. This was even the case in transgenic mice expressing clearly higher HMF sulphation activity in liver and kidney than humans.

Keywords: Nephrotoxicity; Hepatotoxicity; 5-Hydroxymethylfurfural; 5-Sulphooxymethylfurfural; Human sulphotransferases 1A1 and 1A2

The inhibition by di(2-ethylhexyl)-phthalate of erg-mediated K⁺ current in pituitary tumor (GH₃) cells by Sheng-Nan Wu; Wei-Hsin Yang; Chia-Chen Yeh; Hsien-Ching Huang (pp. 713-723).

DEHP (bis(2-ethylhexyl)-phthalate) known to be an endocrine-disrupting chemical is a widely used phthalate. Little information regarding the effects of phthalate esters on ion currents is available. In this study, the effects of DEHP and other phthalate esters (DBEP: di(2-butoxyethyl)-phthalate and DMGP: di(2-methylglycol)-phthalate) on ion currents were investigated in pituitary GH₃ cells. Hyperpolarization-elicited K⁺ currents in GH₃ cells bathed in high-K⁺, Ca²⁺-free solution were examined to evaluate the effects of DEHP, DBEP, and DMGP on the ether-à-go–go-related-gene (erg) K⁺ current (I _K(erg)). Addition of DEHP to GH₃ cells suppressed the amplitude of I _K(erg) in a concentration-dependent manner with an IC₅₀ value of 16.3 μM. With a two-pulse protocol, addition of DEHP shifted the activation curve of I _K(erg) to a depolarized potential by approximately 10 mV with no change in the rate of I _K(erg) deactivation. This compound did not have any effects on delayed rectifier K⁺ current in GH₃ cells, while 4-aminopyridine-3-methanol (100 μM) suppressed this current significantly. DBEP (30 μM) had little or no effect on I _K(erg), while DMGP (30 μM) slightly reduced it. In inside-out configuration, DEHP (30 μM) applied to the bath slightly reduced the activity of large-conductance Ca²⁺-activated K⁺ channels. DEHP (30 μM) increased the frequency of spontaneous action potentials (APs); however, this compound at the same concentration had no effect on AP firing in KCNH2 siRNA-transfected GH₃ cells. The effects described herein can contribute to their actions on functional activity of endocrine or neuroendocrine cells if similar results are found in vivo.

Keywords: di(2-ethylhexyl)-phthalate; GH₃ cells; erg current; K⁺ current

Dominant lethal mutations of topoisomerase II inhibitors etoposide and merbarone in male mice: a mechanistic study by Sabry M. Attia (pp. 725-731).

Two topoisomerase II inhibitors, etoposide and merbarone, were tested for the induction of dominant lethal mutations in male mice. Etoposide was administered at a dosage of 30 or 60 mg/kg. Merbarone was administered at a dosage of 40 or 80 mg/kg. These males were mated at weekly intervals to virgin females for 6 weeks. In the present experiments, regardless of the agent, spermatids appeared to be the most sensitive germ-cell stage to dominant lethal induction. Etoposide and merbarone clearly induced dominant lethal mutations in the early spermatid stage only with the highest tested doses. The mutagenic effects were also directly correlated with reactive oxygen species accumulation as an obvious increase in 2′,7′-dichlorofluorescein fluorescence level was noted in the sperm of animals treated with higher doses of etoposide and merbarone. Treatment of male mice with N-acetylcysteine significantly protected mice from etoposide- and merbarone-induced dominant lethality. Moreover, N-acetylcysteine treatment had no antagonizing effect on etoposide- and merbarone-induced topoisomerase II inhibition. Overall, this study provides for the first time that etoposide and merbarone induce dominant lethal mutations in the early spermatid stage through a mechanism that involves increases in oxidative stress. The demonstrated mutagenicity profile of etoposide and merbarone may support further development of effective chemotherapy with less mutagenicity.

Keywords: Chemotherapy; Mutagenicity; Topoisomerase II; Reactive oxygen species

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA) by Anna Sonnenburg; Varun Ahuja; Maximilian Schreiner; Thomas Platzek; Ralf Stahlmann (pp. 733-740).

Certain textile disperse dyes are known to cause allergic reactions of the human skin. Here, we examined 8 disperse dyes and 7 products of azo-cleavage of these dyes in an in vitro assay. We used the loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells for combined testing of the sensitizing and irritative properties of these substances. The obtained data were compared to data generated in a modified version of the local lymph node assay by our working group. Disperse Blue 1 (DB1), p-nitroaniline (pNA) and p-aminoacetanilide (AAA) showed no sensitizing potential under our experimental conditions. Disperse Blue 124 (DB124), Disperse Yellow 3 (DY3), Disperse Orange 37/76 (DO37), Disperse Blue 106 (DB106), Disperse Red 1 (DR1), 2-amino-p-cresol (ApC), Disperse Orange 3 (DO3) and 2,6-dichloro-4-nitroaniline (DCh) were categorized as extreme sensitizers. Para-phenylenediamine (pPD) was categorized as strong sensitizer, and 2-amino-5-nitrothiazole (ANT) and 2-(N-ethylanilino)-ethanol (EAE) as weak sensitizers. All dyes, except for DB1, and ApC turned out to be strong irritants. DB1, ANT and DCh showed only weak irritative potential. PPD, pNA, EAE and AAA did not show any irritative effect at the concentration range tested. These results correlate with data derived from the modified version of LLNA and human data. Therefore, the LCSA represents a suitable test system to simultaneously analyse two crucial properties of substances relevant for allergy induction.

Keywords: Disperse dyes; Sensitizing potential; In vitro sensitization assay; LCSA

Polyphyllin D induces apoptosis in human erythrocytes through Ca²⁺ rise and membrane permeabilization by Minghui Gao; K. L. Cheung; Irene P. Lau; W. S. Yu; K. P. Fung; Biao Yu; J. F. Loo; S. K. Kong (pp. 741-752).

Polyphyllin D (PD) is a potent anticancer agent isolated from a traditional medicinal herb Paris polyphylla that has been used in China for many years to treat cancer. PD is not a substrate of p-glycoprotein, and it can bypass the multi-drug resistance in cancer cell line R-HepG2. However, the effect of PD on the induction of cell death in human erythrocytes remains unknown. Given that PD is a small molecule that can depolarize the mitochondrial membrane potential and release apoptosis-inducing factor (AIF) in isolated mitochondria, we hypothesized that the apoptogenic effect of PD in human erythrocytes devoid of mitochondria would be minimal. This study therefore tried to evaluate the in vitro effect of PD on hemolysis and apoptosis in human erythrocytes. Apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, after PD treatment was determined by flow cytometry and confocal microscopy for the phosphatidyl-serine externalization and other apoptosis feature events. False to our prediction, PD caused hemolysis and eryptosis/erythroptosis in human RBCs. Mechanistically, elevation in the cytosolic Ca²⁺ ion level seems to be a key but not the only mediator in the PD-mediated eryptosis/erythroptosis because depletion of the external Ca²⁺ could not eliminate the PD effect. Also, PD was able to permeabilize the membrane of RBC ghosts in a way similar to digitonin. Taken together, we report here for the first time the toxicity of PD in human RBCs as well as its underlying mechanism for the hemolysis and eryptosis/erythroptosis.

Keywords: Polyphyllin D; Eryptosis; Erythroptosis; Erythrocytes; Caspase-3; Ca²⁺

Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase, glycolysis, and oxidative phosphorylation by Alvaro Marín-Hernández; Juan Carlos Gallardo-Pérez; Sayra Y. López-Ramírez; Jorge Donato García-García; José Salud Rodríguez-Zavala; Lena Ruiz-Ramírez; Isabel Gracia-Mora; Alejandro Zentella-Dehesa; Marcela Sosa-Garrocho; Marina Macías-Silva; Rafael Moreno-Sánchez; Sara Rodríguez-Enríquez (pp. 753-766).

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC₅₀ = 0.74–6.7 μM) was more effective to inhibit 24–72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC₅₀ = 45–100 μM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3–21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 μM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.

Keywords: Bromopyruvate; Casiopeina; Cisplatin; Fast-growing tumor cells; Glycolysis; Hexokinase

Kinetics of inhibition of soluble peripheral nerve esterases by PMSF: a non-stable compound that potentiates the organophosphorus-induced delayed neurotoxicity by Jorge Estévez; José Barril; Eugenio Vilanova (pp. 767-777).

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus carbamates and sulfonyl fluorides) is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work, kinetic data were obtained for phenylmethylsulfonyl fluoride (PMSF) tested at different concentrations incubated for up to 3 h with soluble fraction of chicken peripheral nerve. PMSF is a protease and esterase inhibitor causing protection or potentiation of the organophosphorus-induced delayed neuropathy and is unstable in water solution. The target of the promotion effect was proposed to be a soluble esterase not yet identified. A kinetic model equation was deduced assuming a multienzymatic system with three different molecular phenomena occurring simultaneously: (1) inhibition, (2) spontaneous chemical hydrolysis of the inhibitor and (3) ongoing inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied for analyzing the experimental data. The best-fitting model is compatible with a resistant component (16.5–18%) and two sensitive enzymatic entities (both 41%). The corresponding second-order rate constants of inhibition (ki = 12.04 × 10⁻² and 0.54 × 10⁻² μM⁻¹ min⁻¹, respectively) and the chemical hydrolysis constant of PMSF (kh = 0.0919 min⁻¹) were simultaneously estimated. These parameters were similar to those deduced in fixed-time inhibition experiments. The consistency of results in both experiments was considered an internal validation of the methodology. The results were also consistent with a significant ongoing inhibition. The proportion of enzymatic components showed in this work is similar to those previously observed in inhibition experiments with mipafox, S9B and paraoxon, demonstrating that this kinetic approach gives consistent results in complex enzymatic systems.

Keywords: Esterases; Kinetics; Organophosphorus; Neurotoxicity; Neuropathy; Phenylmethylsulfonyl fluoride; PMSF; OPIDP; Promotion; Potentiation

Reversible aberration of neurogenesis targeting late-stage progenitor cells in the hippocampal dentate gyrus of rat offspring after maternal exposure to acrylamide by Bunichiro Ogawa; Liyun Wang; Takumi Ohishi; Eriko Taniai; Hirotoshi Akane; Kazuhiko Suzuki; Kunitoshi Mitsumori; Makoto Shibutani (pp. 779-790).

We have recently shown that maternal exposure to acrylamide (AA) impaired neurogenesis in rat offspring measured by the increase in interneurons producing reelin, a molecule regulating migration and correct positioning of developing neurons, in the hippocampal dentate gyrus. To clarify the cellular target of AA on hippocampal neurogenesis and its reversibility after maternal exposure, pregnant Sprague–Dawley rats were given drinking water containing AA at 0, 4, 20, 100 ppm on day 10 of pregnancy through day 21 after delivery on weaning. Male offspring were examined immunohistochemically on postnatal day (PND) 21 and PND 77. For comparison, male pups of direct AA-injection control during lactation (50 mg/kg body weight, intraperitoneally, 3 times/week) were also examined. On PND 21, maternal AA-exposure decreased progenitor cell proliferation in the subgranular zone (SGZ) from 20 ppm accompanied with increased density of reelin-producing interneurons and NeuN-expressing mature neurons within the hilus at 100 ppm, similar to the direct AA-injection control. In the SGZ examined at 100 ppm, cellular populations immunoexpressing doublecortin or dihydropyrimidinase-like 3, suggesting postmitotic immature granule cells, were decreased. On PND 77, the SGZ cell proliferation and reelin-producing interneuron density recovered, while the hilar mature neurons sustained to increase from 20 ppm, similar to the direct AA-injection control. Thus, developmental exposure to AA reversibly affects hippocampal neurogenesis targeting the proliferation of type-3 progenitor cells resulting in a decrease in immature granule cells in rats. A sustained increase in hilar mature neurons could be the signature of the developmental effect of AA.

Keywords: Acrylamide; Neuronal development; Hippocampal dentate gyrus; Impaired neurogenesis; Immunohistochemistry; Reversibility

Carcinogenicity of ortho-phenylenediamine dihydrochloride in rats and mice by two-year drinking water treatment by Michiharu Matsumoto; Masaaki Suzuki; Hirokazu Kano; Shigetoshi Aiso; Kazunori Yamazaki; Shoji Fukushima (pp. 791-804).

The carcinogenicity of ortho-phenylenediamine (o-PD) was examined by administrating o-phenylenediamine dihydrochloride (o-PD2HCl) in dinking water to groups of 50 F344/DuCrj rats and 50 Crj:BDF₁ mice of both sexes for 2 years. The drinking water concentration of o-PD2HCl was 0, 500, 1,000 or 2,000 ppm (wt/wt) for male rats; 0, 250, 500 or 1,000 ppm for female rats; 0, 500, 1,000 or 2,000 ppm for male mice; and 0, 1,000, 2,000 or 4,000 ppm for female mice. Two-year administration of o-PD2HCl produced a dose-dependent increase in the incidences of hepatocellular adenomas and carcinomas in rats of both sexes and in female mice, and hepatocellular adenomas in male mice. In mice, the incidences of hepatocellular adenomas were increased at the lowest dose used in both males and females. Metastasis from hepatocellular carcinomas of rats occurred predominantly in the lung. Incidences of transitional cell papillomas and carcinomas in the urinary bladder were noted in male rats administered 2,000 ppm, together with an increased incidence of papillary and/or nodular hyperplasia of transitional epithelium. In mice, the incidence of papillary adenomas in the gall bladder, which is rare in mice, was increased in both males and females administered 2,000 ppm. Thus, o-PD is carcinogenic in two species, i.e., rats and mice of both sexes.

Keywords: ortho-Phenylenediamine; Carcinogenicity; Rodents; Liver; Gall bladder; Urinary bladder

Cytotoxic and DNA-damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells by Verena J. Koller; Maria Fürhacker; Armen Nersesyan; Miroslav Mišík; Maria Eisenbauer; Siegfried Knasmueller (pp. 805-813).

Glyphosate (G) is the largest selling herbicide worldwide; the most common formulations (Roundup, R) contain polyoxyethyleneamine as main surfactant. Recent findings indicate that G exposure may cause DNA damage and cancer in humans. Aim of this investigation was to study the cytotoxic and genotoxic properties of G and R (UltraMax) in a buccal epithelial cell line (TR146), as workers are exposed via inhalation to the herbicide. R induced acute cytotoxic effects at concentrations >40 mg/l after 20 min, which were due to membrane damage and impairment of mitochondrial functions. With G, increased release of extracellular lactate dehydrogenase indicative for membrane damage was observed at doses >80 mg/l. Both G and R induced DNA migration in single-cell gel electrophoresis assays at doses >20 mg/l. Furthermore, an increase of nuclear aberrations that reflect DNA damage was observed. The frequencies of micronuclei and nuclear buds were elevated after 20-min exposure to 10–20 mg/l, while nucleoplasmatic bridges were only enhanced by R at the highest dose (20 mg/l). R was under all conditions more active than its active principle (G). Comparisons with results of earlier studies with lymphocytes and cells from internal organs indicate that epithelial cells are more susceptible to the cytotoxic and DNA-damaging properties of the herbicide and its formulation. Since we found genotoxic effects after short exposure to concentrations that correspond to a 450-fold dilution of spraying used in agriculture, our findings indicate that inhalation may cause DNA damage in exposed individuals.

Keywords: Glyphosate; Roundup; Cytotoxic; Comet assay; Micronuclei; Buccal cells

Influence of a fat-rich diet, folic acid supplementation and a human-relevant concentration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on the induction of preneoplastic lesions in the rat colon by Petra Nicken; Nicole Brauer; Alfonso Lampen; Pablo Steinberg (pp. 815-821).

In the present study, the effect of three controversially discussed risk factors for colorectal cancer, a fat-rich diet (16% raw fat content), dietary folic acid supplementation (50 mg folic acid/kg lab chow) and a human-relevant concentration (0.1 ppm) of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), either alone or in combination, on the induction of aberrant crypt foci (ACF) in the colon of male Fischer 344 rats was analyzed. The mean number of ACF per rat in the case of the four groups fed a fat-rich diet tended to be higher than that of the four groups being fed a standard diet. However, the increase in the mean number of ACF per rat only reached statistical significance in the case of the rats receiving a fat-rich lab chow supplemented with 50 mg/kg folic acid. Moreover, a concentration of 0.1 ppm PhIP per se, either in the standard or in the fat-rich lab chow, did not lead to an increase in the mean number of ACF per rat. In conclusion, the present study provides additional evidence for a colon cancer promoting effect of folic acid supplementation when rodents are fed the compound in supraphysiological concentrations.

Keywords: Aberrant crypt foci; Colorectal cancer; Fat-rich diet; Folic acid; Heterocyclic aromatic amines

Erratum to: Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway by Tsung-Ho Ying; Shun-Fa Yang; Su-Ju Tsai; Shu-Ching Hsieh; Yi-Chang Huang; Da-Tian Bau; Yi-Hsien Hsieh (pp. 823-823).

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Archives of Toxicology (v.86, #5)