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Archives of Toxicology (v.80, #8)

Cytotoxicity of dental composite (co)monomers and the amalgam component Hg²⁺ in human gingival fibroblasts by Franz-Xaver Reichl; Sabine Simon; Magalie Esters; Mario Seiss; Kai Kehe; Norbert Kleinsasser; Reinhard Hickel (pp. 465-472).

Unpolymerized resin (co)monomers or mercury (Hg) can be released from restorative dental materials (e.g. composites and amalgam). They can diffuse into the tooth pulp or the gingiva. They can also reach the gingiva and organs by the circulating blood after the uptake from swallowed saliva. The cytotoxicity of dental composite components hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (Bis-GMA) as well as the amalgam component Hg²⁺ (as HgCl₂) and methyl mercury chloride (MeHgCl) was investigated on human gingival fibroblasts (HGFs) at two time intervals. To test the cytotoxicity of substances, the bromodeoxyuridine (BrdU) assay and the lactate dehydrogenase (LDH) assay were used. The test substances were added in various concentrations and cells were incubated for 24 or 48 h. The EC₅₀ values were obtained as half-maximum-effect concentrations from fitted curves. Following EC₅₀ values were found [BrdU: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 8.860 (0.440)/6.600(0.630), TEGDMA 1.810(0.130)/1.220(0.130), UDMA 0.120(0.010)/0.140(0.010), BisGMA 0.060(0.004)/0.040(0.002), HgCl₂ 0.015(0.001)/0.050(0.006), and MeHgCl 0.004(0.001)/0.005(0.001). Following EC₅₀ values were found [LDH: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 9.490(0.300)/7.890(1.230), TEGDMA 2.300(0.470)/1.950(0.310), UDMA 0.200(0.007)/0.100(0.007), BisGMA 0.070(0.005)/0.100(0.002), and MeHgCl 0.014(0.006)/0.010(0.003). In both assays, the following range of increased toxicity was found for composite components (24 and 48 h): HEMA < TEGDMA < UDMA < BisGMA. In both assays, MeHgCl was the most toxic substance. In the BrdU assay, Hg²⁺ was about fourfold less toxic than MeHgCl but Hg²⁺ was about fourfold more toxic than BisGMA. In the BrdU test, a significantly (P<0.05) decreased toxicity was observed for Hg²⁺ at 48 h, compared to the 24 h Hg²⁺-exposure. A time depending decreased toxicity was observed only for Hg²⁺ which can then reach the toxic level of BisGMA.

Keywords: Cytotoxicity; Lactate dehydrogenase assay; Bromodeoxyuridine assay; Comonomers; Amalgam; Gingival cells

Effects of depleted uranium after short-term exposure on vitamin D metabolism in rat by E. Tissandie; Y. Guéguen; J. M. A. Lobaccaro; F. Paquet; J. Aigueperse; M. Souidi (pp. 473-480).

Uranium is a natural radioactive heavy metal. Its toxicity has been demonstrated for different organs, including bone, kidney, liver and brain. Effects of an acute contamination by depleted uranium (DU) were investigated in vivo on vitamin D₃ biosynthetic pathway. Rats received an intragastric administration of DU (204 mg/kg) and various parameters were studied either on day 1 or day 3 after contamination. Cytochrome P450 (CYP27A1, CYP2R1, CYP27B1, CYP24A1) enzymes involved in vitamin D metabolism and two vitamin D₃-target genes (ECaC1, CaBP-D9K) were assessed by real time RT-PCR in liver and kidneys. CYP27A1 activity was measured in liver and vitamin D and parathyroid hormone (PTH) level were measured in plasma. In acute treated-rats, vitamin D level was increased by 62% and decreased by 68% in plasma, respectively at day 1 and at day 3, which paralleled with a concomitant decrease of PTH level (90%) at day 3. In liver, cyp2r1 mRNA level was increased at day 3. Cyp27a1 activity decreased at day 1 and increased markedly at day 3. In kidney, cyp27b1 mRNA was increased at days 1 and 3 (11- and 4-fold respectively). Moreover, ecac1 and cabp-d9k mRNA levels were increased at day 1 and decreased at day 3. This work shows for the first time that DU acute contamination modulates both activity and expression of CYP enzymes involved in vitamin D metabolism in liver and kidney, and consequently affects vitamin D target genes levels.

Keywords: Depleted uranium; Cytochrome P450; Vitamin D₃

Determination of ochratoxin A by polyclonal antibodies based sensitive time-resolved fluoroimmunoassay by Biao Huang; Wenyi Tao; Jin Shi; Luhong Tang; Jian Jin (pp. 481-485).

Using indirect competitive time-resolved fluoroimmunoassay (TRFIA), a rapid, highly selective and extremely sensitive method has been established for the determination of ochratoxin A (OA). Tests can be performed in a 96-well microplate using the toxin-specific polyclonal antibodies, obtained from rabbits immunized with ochratoxin A-keyhole limpet’s hemocyanin (OA–KLH). In indirect TRFIA format, ochratoxin A-bovine serum albumin conjugate (OA–BSA) is coated onto the microtitre plate and incubated with standard toxin (samples) and anti-OA antibody. A goat anti-rabbit IgG Eu³⁺ conjugate is used to enable the detection. The suitability of the assay for quantification of OA is also studied and samples are determined by OA-TRFIA using autoDELFIA1235 system. The results show that the polyclonal antibodies can be used at a dilution exceeding 1:8,000 and the OA detection limit is 0.02 μg/l for indirect competitive TRFIA formats. The 80, 50, and 20% inhibition binding effect dose (ED80, ED50, ED20) of OA were 0.195, 1.018, and 5.314 μg/l, respectively. The assay ranges from 0.02 to 400 μg/l. The cross reactivity with ochratoxin B is 5.6% and antibodies do not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OA-TRFIA are 2.6 and 5.2%, respectively. The mean recoveries from the OA-free cereals spiked with 1–200 μg of OA/kg of cereals sample were 95.8%. Both OA-TRFIA and OA-ELISA tests are applied for the quantitative measurement of OA in the same cereals, and the coefficient of correlation is 0.912. The results show that the novel TRFIA method can be applied to detect the OA contamination in cereals. It provides very high sensitivity and optimal range and will be useful to screen OA contamination easily, simply, and economically when the number of samples is large.

Keywords: Time-resolved fluoroimmunoassay; Ochratoxin A; Polyclonal antibodies; Mycotoxin

The role of glutathione on the cytotoxic effects and cellular uptake of diphenylarsinic acid, a degradation product of chemical warfare agents by Takafumi Ochi; Kenji Kinoshita; Toshihide Suzuki; Kouichi Miyazaki; Ayano Noguchi; Toshikazu Kaise (pp. 486-491).

The mechanism underlying enhancement of the cytotoxic effects of diphenylarsinic acid (DPAA) by sulfhydryl (SH) compounds, such as glutathione (GSH) and dimercaptopropane sulfonate (DMPS), was investigated in terms of not only the effects of SH compounds on DPAA uptake by cells, but also the cytotoxic effects of the GSH adduct of DPAA, DPA-GS. In addition, the cytotoxic effects of DPA-GS and cellular uptake were investigated in conjunction with the effects of GSH depletion. Cells took up DPAA in a time- and temperature-dependent manner for up to 2 h, then the uptake leveled off for 6 h. Arsenic species other than DPAA were not detected in the cells. The presence of GSH and DMPS did not influence the rate of uptake of DPAA by the cells. By contrast, when the cytotoxic potential of DPA-GS was compared with that of DPAA, DPA-GS was about 1,000 times more toxic than DPAA, suggesting that enhancement of DPAA toxicity by SH compounds might be due to the formation of adducts in the culture medium. The cytotoxic effects of DPA-GS were suppressed markedly by the presence of GSH and DMPS, and the suppression was attributed to an inhibition of more than 90% by the SH compounds of DPA-GS uptake. Depletion of cell GSH enhanced the cytotoxic effects of DPA-GS by two to three times and the enhancement attributed to an increased cellular uptake of DPA-GS. These results suggest that GSH plays a role in regulating the formation of DPA-GS and cellular uptake.

Keywords: Diphenylarsinic acid (DPAA); Glutathione adduct of DPAA (DPA-GS); Cellular uptake

Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals by Katharina Krüger; Janina Gruner; Michael Madeja; Louise M. Hartmann; Alfred V. Hirner; Norbert Binding; Ulrich Mußhoff (pp. 492-501).

Pentavalent and trivalent organoarsenic compounds belong to the major metabolites of inorganic arsenicals detected in humans. Recently, the question was raised whether the organic arsenicals represent metabolites of a detoxification process or methylated species with deleterious biological effects. In this study, the effects of trivalent arsenite (AsO₃ ³⁻; iA^III), the pentavalent organoarsenic compounds monomethylarsonic acid (CH₃AsO(OH)₂; MMA^V) and dimethylarsinic acid ((CH₃)₂AsO(OH); DMA^V) and the trivalent compounds monomethylarsonous acid (CH₃As(OH)₂, MMA^III) and dimethylarsinous acid ((CH₃)₂As(OH); DMA^III) were tested on glutamate receptors and on voltage-operated potassium and sodium channels heterologously expressed in Xenopus oocytes. Membrane currents of ion channels were measured by conventional two-electrode voltage-clamp techniques. The effects of arsenite were tested in concentrations of 1–1,000 μmol/l and the organic arsenical compounds were tested in concentrations of 0.1–100 μmol/l. We found no significant effects on voltage-operated ion channels; however, the arsenicals exert different effects on glutamate receptors. While MMA^V and MMA^III significantly enhanced ion currents through N-methyl-d-aspartate (NMDA) receptor ion channels with threshold concentrations <10 μmol/l, DMA^V and DMA^III significantly reduced NMDA-receptor mediated responses with threshold concentrations <0.1 μmol/l; iA^III had no effects on glutamate receptors of the NMDA type. MMA^III and DMA^V significantly reduced ion currents through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor ion channels with threshold concentrations <10 μmol/l (MMA^III) and <1 μmol/l (DMA^V). MMA^V and iA^III had no significant effects on glutamate receptors of the AMPA type. The effects of MMA^V, MMA^III, DMA^V and DMA^IIIon glutamate receptors point to a neurotoxic potential of these substances.

Keywords: Xenopus oocyte; Membrane currents; Potassium channels; Sodium channels; Arsenite

Anticonvulsant effects of GABA_A modulators microinfused into area tempestas or substantia nigra in rats exposed to soman by Trond Myhrer; Nga H. T. Nguyen; Siri Enger; Pål Aas (pp. 502-507).

Enhancement of GABAergic neurotransmission has anticonvulsant effects against nerve agent-induced seizures. However, systemic administration of drugs with GABA_A agonist-like effects does not differentiate well between their anticonvulsant impact. In the present study, GABA_A modulating drugs (1 μl) were microinfused bilaterally into the seizure controlling substrates, substantia nigra (SN) or area tempestas (AT), of rats subjected to seizures induced systemically by soman (100 μg/kg). The results showed that infusion of ethanol (0.47 μmol) and propofol (20 μg) in both SN and AT had anticonvulsant effects (prevention of seizures or increased latency to seizures). Anticonvulsant effects were also obtained when muscimol (120 ng) was infused into AT or when diazepam (5 μg) was infused into SN. Pentobarbital (50 μg) did not attenuate soman-elicited seizures in any of the injection sites. Results from control experiments showed that the effects from the microinfusions were site-specific, and that the absence of effects of pentobarbital was not a result of too low dose of the drug. The microinfusion technique may allow a more detailed examination of anticonvulsant properties of drugs than by the use of systemic administration.

Keywords: GABA_Amodulators; Substantia nigra; Area tempestas; Soman seizures; Anticonvulsant effects; Rats

Toxicity of jet fuel aliphatic and aromatic hydrocarbon mixtures on human epidermal Keratinocytes: evaluation based on in vitro cytotoxicity and interleukin-8 release by Jen-Hung Yang; Chia-Hue Lee; Nancy A. Monteiro-Riviere; Jim E. Riviere; Chau-Loong Tsang; Chi-Chung Chou (pp. 508-523).

Jet fuels are complex mixtures of aliphatic (ALI) and aromatic (ARO) hydrocarbons that vary significantly in individual cytotoxicity and proinflammatory activity in human epidermal keratinocytes (HEK). In order to delineate the toxicological interactions among individual hydrocarbons in a mixture and their contributions to cutaneous toxicity, nine ALI and five ARO hydrocarbons were each divided into five (high/medium/low cytotoxic and strong/weak IL-8 induction) groups and intra/inter-mixed to assess for their mixture effects on HEK mortality and IL-8 release. Addition of single hydrocarbon to JP-8 fuel was also evaluated for their changes in fuel dermatotoxicity. The results indicated that when hydrocarbons were mixed, HEK mortality and IL-8 release were not all predictable by their individual ability affecting these two parameters. The lowest HEK mortality (7%) and the highest IL-8 production were induced with mixtures including high cytotoxic and weak IL-8 inductive ARO hydrocarbons. Antagonistic reactions not consistently correlated with ALI carbon chain length and ARO structure were evident and carried different weight in the overall mixture toxicities. Single addition of benzene, toluene, xylene or ethylbenzene for up to tenfold in JP-8 did not increase HEK mortality while single addition of ALI hydrocarbons exhibited dose-related differential response in IL-8. In an all ALI environment, no single hydrocarbon is the dominating factor in the determination of HEK cytotoxicity while deletion of hexadecane resulted in a 2.5-fold increase in IL-8 production. Overall, decane, undecane and dodecane were the major hydrocarbons associated with high cytotoxicity while tetradecane, pentadecane and hexadecane were those which had the greatest buffering effect attenuating dermatotoxicity. The mixture effects must be considered when evaluating jet fuel toxicity to HEK.

Keywords: Hydrocarbon mixtures; Jet fuels; Keratinocyte; Cytotoxicity; Interleukin-8

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro by Ying Li; Zhihong Liu; Xiaohua Guo; Jian Shu; Zhaohong Chen; Leishi Li (pp. 524-532).

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.

Keywords: Aristolochic acid I; Proximal tubular epithelial cells; DNA damage; Cell cycle arrest; Nephrotoxicity

Gene expression analysis in mice liver on hepatocarcinogenesis by flumequine by Yoko Kashida; Akiko Takahashi; Mitsuyoshi Moto; Miwa Okamura; Masako Muguruma; Meilan Jin; Katsuhiko Arai; Kunitoshi Mitsumori (pp. 533-539).

mRNA expression profiles in the liver from mice treated with flumequine (FL) were analyzed in order to elucidate the mechanism of its tumor-promoting effect. The liver from a C3H/He mouse that received a diet containing 4,000 ppm of FL for 4 weeks was examined by cDNA microarray in comparison with an untreated mouse. Furthermore, to obtain a more comprehensive sequence, time-course changes in selected genes were determined by real-time RT-PCR. Microarray analysis revealed 15 upregulated and 9 downregulated genes in an FL-treated mouse. The upregulated genes included signal transducers and cell cycle regulators. In addition, the levels of stress response genes, particularly glutathione S-transferase (GST) α and GSTμ, were very high, indicating the generation of oxidative stress. On the other hand, the downregulated genes included phase I metabolic enzymes, such as cytochrome P450 (CYPs) enzymes, and apoptosis-associated proteins. These changes were confirmed by quantitative RT-PCR and were generally consistent with each other. Time-course observations revealed consistent results, particularly with regard to GSTα, GSTμ, ERK5, and CYP2E1. In addition, the expression of 8-oxoguanine DNA glycosylase 1 (OGG1) was increased in a time-dependent manner. These results suggest the possibility that responses against oxidative stress may play a major role in hepatocarcinogenesis by FL in mice.

Keywords: Flumequine; Hepatocarcinogenesis; Oxidative stress; Tumor-promoting effect

Influence of fuel properties, nitrogen oxides, and exhaust treatment by an oxidation catalytic converter on the mutagenicity of diesel engine emissions by Jürgen Bünger; Jürgen Krahl; Andreas Weigel; Olaf Schröder; Thomas Brüning; Michael Müller; Ernst Hallier; Götz Westphal (pp. 540-546).

Particle emissions of diesel engines (DEP) content polycyclic aromatic hydrocarbons (PAH) these compounds cause a strong mutagenicity of solvent extracts of DEP. We investigated the influence of fuel properties, nitrogen oxides (NO x), and an oxidation catalytic converter (OCC) on the mutagenic effects of DEP. The engine was fuelled with common diesel fuel (DF), low-sulphur diesel fuel (LSDF), rapeseed oil methyl ester (RME), and soybean oil methyl ester (SME) and run at five different load modes in two series with and without installation of an OCC in the exhaust pipe. Particles from the cooled and diluted exhaust were sampled onto glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The mutagenicity of the extracts was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Without OCC the number of revertant colonies was lower in extracts of LSDF than in extracts of DF. The lowest numbers of revertant colonies were induced by the plant oil derived fuels. In three load modes, operation with the OCC led to a reduction of the mutagenicity. However, direct mutagenic effects under heavy duty conditions (load mode A) were significantly increased for RME (TA98, TA100) and SME (TA98). A consistent but not significant increase in direct mutagenicity was observed for DF and LSDF at load mode A, and for DF at idling (load mode E) when emissions were treated with the OCC. These results raise concern over the use of oxidation catalytic converters with diesel engines. We hypothesise that the OCC increases formation of direct acting mutagens under certain conditions by the reaction of NO x with PAH resulting in the formation of nitrated-PAH. Most of these compounds are powerful direct acting mutagens.

Keywords: Nitrogen oxides; Diesel engine particles; Oxidation catalytic converter; Nitrated polycyclic aromatic hydrocarbons; Salmonella typhimurium/mammalian microsome assay

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Archives of Toxicology (v.80, #8)