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Archives of Toxicology (v.74, #8)

No Title by P. Janning; U.S. Schuhmacher; A. Upmeier; P. Diel; H. Michna; G.H. Degen; H.M. Bolt (pp. 421-430).

Female DA/Han rats were given the phytoestrogen daidzein, either intravenously (10 mg/kg b.w.) or orally by gavage (10 or 100 mg/kg b.w.). The plasma concentration–time curve determined after i.v. administration of daidzein was fitted to a triexponential model, resulting in a final half-life (γ-phase) of approximately 4 h. The oral bioavailability of 10 mg daidzein/kg was 9.7%, while that of 100 mg/kg was 2.2%; the higher dose (100 mg/kg) was apparently absorbed to a four- to fivefold lower extent than the smaller dose. The plasma concentration–time curves after oral administration of daidzein to female DA/Han rats revealed pronounced interindividual differences and multiple peaks, pointing to extensive enterohepatic circulation and/or protracted absorption from the gastrointestinal tract. As shown in a separate experiment with bile duct-cannulated rats, daidzein (i.p. 10 mg/kg b.w.) is efficiently excreted with bile: glucuronide/sulfate metabolites amounting to approximately 30% of the dose in 8 h. Conjugates were also the main circulating metabolites upon i.v. or gavage administration of daidzein, indicating efficient phase II metabolism in female DA/Han rats. Since only few data have been published on tissue levels of isoflavones, their concentrations were measured in various organs and compared to plasma levels determined at the time the animals were killed, with one exception 32 or 48 h after rats had received a single dose of daidzein (i.v. or per os). As expected, the daidzein concentrations depended upon dose and administration route. Despite notable differences in the absolute amounts of total daidzein (free plus hydrolyzed conjugates), the levels were usually three- to fivefold higher in liver and kidney than in plasma; in most samples of uteri, the concentrations were similar, or up to twofold higher, than the respective plasma levels. These data point to an uptake and storage of isoflavones and metabolites in tissues. Experimental toxicokinetics appear to be a relevant subject that should be integrated into assessments of toxicological data for endocrine modulators.

Keywords: Daidzein Environmental estrogens Toxicokinetics Enterohepatic circulation

No Title by Andreas Upmeier; Gisela H. Degen; Patrick Diel; Horst Michna; Hermann M. Bolt (pp. 431-436).

Bisphenol A [BPA; 2,2-bis-(4-hydroxyphenyl)-propane] is a monomer used in the manufacture of resins with a wide range of applications, e.g. plastic coatings in the food packaging industry. BPA has been shown to have a weak oestrogenic activity in vitro and in vivo. Despite its low oestrogenic potency there is concern that, as a consequence of slow clearance, BPA might reach biologically significant levels in humans and animals exposed to environmental levels. To address this concern, we assessed the kinetic behaviour of BPA in female DA/Han rats. Groups of female rats received 10 mg BPA/kg body weight intravenously or 10 or 100 mg BPA/kg body weight orally (by gavage). Blood samples were collected at different time-points and plasma was prepared. Free BPA in the samples was isolated by fluid-fluid extraction. BPA was measured by GC-MS which allowed the reliable determination of BPA concentrations as low as approximately 10 ng/ml plasma. Immediately after i.v. administration, the BPA plasma concentration was in the range of about 15 µg/ml and decreased rapidly within the first hour (to 700 ng/ml). The levels declined further (100 ng/ml at 2 h), and after 24 h the analytical detection limit was reached. BPA was detected in plasma as early as 10 min after gavage administration, indicating rapid initial uptake from the gastrointestinal tract. Absorption of BPA was variable. In animals receiving 10 mg/kg, maximal plasma levels were reached after 1.5 h (31 ng/ml) and 6 h (40 ng/ml). In animals receiving 100 mg/kg, plasma levels reached maxima around 30 min (150 ng/ml) and 3 h (134 ng/ml) after administration. After 48 h BPA was at or below the detection limit in both dose groups. Fluctuations in the BPA plasma concentrations over time point to the possibility of enterohepatic recirculation and protracted absorption from the gastrointestinal tract. Using the area under the concentration-time curves (AUCs), low bioavailabilities of 16.4% and 5.6% were calculated for the 10 and 100 mg/kg dose groups, respectively. The toxicokinetic properties of BPA in DA/Han rats are in agreement with the hypothesis of a rapid first-pass elimination by the liver and efficient metabolic clearance of low oral doses. Only excessive doses may lead to bioaccumulation if detoxification pathways are saturated.

Keywords: Bisphenol A Environmental oestrogens Toxicokinetics Enterohepatic circulation

No Title by I. Gut; V. Danielová; J. Holubová; P. Souček; H. Klučková (pp. 437-446).

Cytotoxic effects of cyclophosphamide (CPA), paclitaxel (PCT), and docetaxel (DTX) and their modulation by cytochrome P450 (CYP) metabolism were studied by incubating cell lines L929 and P388D₁ with or without rat liver microsomes. The microsomes themselves were not cytotoxic. P388D₁ cells were more sensitive to CPA, PCT, and DTX than L929 cells. CYP2B1-, CYP3A-, and CYP2E1-induced microsomes effectively oxidized the prodrug CPA to cytotoxic products in 2-h incubation periods. Cytotoxicity of DTX and PCT for P388D₁ cells became apparent 24 h after a 2-h incubation period with the drugs, and their effects were enhanced by CYP2E1 microsomes, but markedly decreased by CYP3A-induced microsomes. DTX and PCT without microsomes caused a dose-related cytotoxicity in P388D₁ and HeLa cells. P388D₁ and HeLa cells did not grow after a 24-h exposure to 1–10 µM DTX, but about 0.1% of cells survived exposure to 1–10 µM PCT. After 4 weeks of multiplication, the surviving P388D₁ cells displayed lower sensitivity to DTX and PCT, but cytotoxicity in HeLa cells was unchanged and their growth ability decreased. In P388D₁ cells, PCT with DTX (0.1, 0.5, 1, 2.5, or 5 µM) showed only additive cytotoxicity, although they reportedly act in different phases of the cell cycle. In P388D₁ cells treated with DTX or PCT, normal mononuclear cells disappeared and the cell diameter increased up to threefold. Mulberry-like nuclei developed, giving rise to multiple nuclei, which were hyper- or hypochromatic. Chromatin condensation in some multiple nuclei and cell shrinkage of some cells fit the definition of apoptosis, but enlargement of the surviving cells and numerous hypochromatic nuclei do not. In conclusion, L929 and P388D₁ cells incubated with microsomes enabled the role of various CYP enzymes in the effect of anticancer drugs to be assessed. The delayed cytotoxicity of DTX and PCT compared to that of CPA was related to their different mode of action. Fluorescent microscopy revealed quantitatively different effects of PCT and DTX on the nuclei, indicating that their mode of action may not be completely identical.

Keywords: Cyclophosphamide Docetaxel Paclitaxel Cytochrome P450 Cytotoxicity Apoptosis Nuclear morphology

No Title by Chung-Ren Jan; Kang-Ju Chou; Kam Chung Lee; Jue-Long Wang; Li-Ling Tseng; Jin-Shiung Cheng; Wei-Chuan Chen (pp. 447-451).

The effect of the phospholipase A₂ inhibitor palmitoyl trifluoromethyl ketone (PACOCF₃) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined using fura-2 as the fluorescent Ca²⁺ indicator. At a concentration of 20 µM, PACOCF₃ did not change basal cytosolic free calcium concentrations ([Ca²⁺]_i), but at concentrations of 50–250 µM PACOCF₃ induced an increase in [Ca²⁺]_i by activating extracellular Ca²⁺ entry which was partly suppressed by 50 µM La³⁺. The effect of PACOCF₃ was abolished by removal of extracellular Ca²⁺. PACOCF₃ (10 µM) enhanced both the peak value and the area under the curve of the [Ca²⁺]_i increase induced by 10 µM ATP and 1 µM bradykinin by potentiating extracellular Ca²⁺ influx without affecting internal Ca²⁺ release. Several other phospholipase A₂ inhibitors had no effect on basal [Ca²⁺]_i or agonist-induced [Ca²⁺]_i increases. Collectively, the results suggest that PACOCF₃ alters Ca²⁺ signaling in renal tubular cells in a manner independent of phospholipase A₂ inhibition.

Keywords: PACOCF3 Calcium, intracellular Fura-2 MDCK cells

No Title by J.Y.C. Ma; M.W. Barger; A.J. Kriech; V. Castranova (pp. 452-459).

Objective: The present study was carried out to characterize the effects of in vitro exposure to paving asphalt fume condensate (AFC) on alveolar macrophage (AM) functions and to monitor acute pulmonary responses to in vivo AFC exposure in rats. Methods: For in vitro studies, rat primary AM cultures were incubated with various concentrations of AFC for 24 h at 37°C. AM-conditioned medium was collected and assayed for lactate dehydrogenase (LDH) as a marker of cytotoxicity. Tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) production were assayed in AM-conditioned medium to monitor AM function. The effect of AFC on chemiluminescence (CL) generated by resting AM or AM in response to zymosan or PMA stimulation was also determined as a marker of AM activity. For in vivo studies, rats received either (1) a single intratracheal (IT) instillation of saline, or 0.1 mg or 0.5 mg AFC and were killed 1 or 3 days later; or (2) IT instillation of saline, or 0.1, 0.5, or 2 mg AFC for three consecutive days and were killed the following day. Differential counts of cells harvested by bronchoalveolar lavage were measured to monitor inflammation. Acellular LDH and protein content in the first lavage fluid were measured to monitor damage. CL generation, TNF-α and IL-1 production by AM were assayed to monitor AM function. Results: In vitro AFC exposure at <200 µg/ml did not induce cytotoxicity, oxidant generation, or IL-1 production by AM, but it did cause a small but significant increase in TNF-α release from AM. In vitro exposure of AM to AFC resulted in a significant decline of CL in response to zymosan or PMA stimulation. The in vivo studies showed that AFC exposure did not induce significant neutrophil infiltration or alter LDH or protein content in acellular lavage samples. Macrophages obtained from AFC-exposed rats did not show significant differences in oxidant production or cytokine secretion at rest or in response to LPS in comparison with control macrophages. Conclusions: These results suggest that: (1) in vitro AFC exposure did not adversely affect cell viability or induce the release of high levels of inflammatory cytokines or oxidants; and (2) exposure of rats to AFC did not cause acute pulmonary inflammation or injury, and did not significantly alter AM functions.

Keywords: Paving asphalt Asphalt fume condensate Alveolar macrophage Pulmonary inflammation Lung injury

No Title by E. Le Prieur; E. Vaz; A. Bion; F. Dionnet; J.-P. Morin (pp. 460-466).

Precision-cut rat lung slices in organotypic culture placed in a biphasic air/liquid system were used for this study. This model allowed pathological as well as cellular and molecular biology investigations to be carried out. Slices were exposed to a continuous flow of diluted diesel exhaust, with a pO₂ adjusted to 20% to avoid hypoxia-induced effects. The exposure system allowed five exhaust concentrations from the same diesel engine to be studied concomitantly, and also allowed the impact of removing the particulate matter using a filter cap on the exposure vials to be evaluated. Lung slices were exposed for 3 or 6 h to whole or filtered diesel exhaust. DNA integrity was characterized by two different techniques: (1) an ELISA for the determination of nucleosomes, and (2) the histochemical TUNEL method. By the TUNEL method, apoptotic cells were detected after a 6-h exposure followed by an incubation period of 18 h in a controlled atmosphere comprising 5% CO₂/95% O₂. Under these conditions, apoptotic nuclei were more frequent in slices exposed to diesel exhaust than in control slices. Cytokine production (tumor necrosis factor α, interleukin-1β) in the culture medium was measured using an ELISA technique. After a 3-h exposure only TNF-α was detected and increased in the culture medium of lung slices exposed to diesel exhaust. Under the same conditions, nucleosome levels in the slices increases in a dose-dependent way. In conclusion, whole diesel exhaust induced an inflammatory response and DNA alterations which were reduced by filtration, thus indicating the important role of the particulate matter in diesel exhaust.

Keywords: Diesel exhausts Lung slices Inflammation Apoptosis Organotypic culture

No Title by Wea-Lung Lin; Chau-Jong Wang; Yu-Ying Tsai; Chuen-Lan Liu; Jin-Ming Hwang; Tsui-Hwa Tseng (pp. 467-472).

Increasing evidence regarding free radical-generating agents and inflammatory processes suggests that accumulation of reactive oxygen species can cause hepatotoxicity. A short-chain analog of lipid hydroperoxide, t-butyl hydroperoxide (t-BHP), can be metabolized to free radical intermediates by cytochrome P-450 in hepatocytes, which in turn can initiate lipid peroxidation, affect cell integrity and result in cell injury. In this study, we used t-BHP to induce hepatotoxicity in vitro and in vivo and determined the antioxidative bioactivity of esculetin, a coumarin compound. Our investigations showed that pretreatment with esculetin (5–20 µg/ml) significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine transaminase (ALT), and also decreased the formation of malondialdehyde (MDA) in primary cultured rat hepatocytes induced by a 30-min treatment with t-BHP. An in vivo study in rats showed that pretreatment with esculetin (i.p.) at concentrations of 0.5 and 5 mg/kg for 5 days before a single i.p. dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of the hepatic enzyme markers (ALT and AST) and reduced oxidative stress in the liver. Histopathological evaluation of the rat livers revealed that esculetin reduced the incidence of liver lesions induced by t-BHP, including hepatocyte swelling, leukocyte infiltration, and necrosis. Based on the results described above, we speculate that esculetin may play a chemopreventive role via reducing oxidative stress in living systems.

Keywords: Esculetin t-Butyl hydroperoxide Hepatotoxicity

No Title by R. Konno; M. Ikeda; K. Yamaguchi; Y. Ueda; A. Niwa (pp. 473-479).

When D-propargylglycine was injected intraperitoneally into mice, polyuria, glycosuria, and aminoaciduria were observed as has been previously reported in rats. The urine of the mice treated with D-propargylglycine contained twice as much protein as that of the control mice. Polyacrylamide gel electrophoresis showed a new protein of approximately 62 kDa in the urine of the D-propargylglycine-treated mice. Protein sequencing revealed that this protein was serum albumin. Since the above-mentioned symptoms suggested dysfunction of the renal proximal tubules, the activity of urinary N-acetyl-β-D-glucosaminidase, a marker enzyme of injury to the proximal tubules, was measured. The urinary enzyme activity was 2.6 times higher in the D-propargylglycine-treated mice than in the control mice. Light- and electron-microscopy showed degenerative and necrotic cells in the straight part of the proximal tubules of the treated mice. However, none of these symptoms was observed in D-propargylglycine-treated mutant mice, lacking D-amino-acid oxidase. These results indicate that D-propargylglycine itself is not nephrotoxic but its metabolite produced by the D-amino-acid oxidase reaction is nephrotoxic and injures proximal tubular cells, resulting in an impairment of the reabsorption of water, glucose, amino acids, and proteins.

Keywords: D-Propargylglycine D-Amino-acid oxidase Mice Proximal tubule Mutant mouse

No Title by G. Lallement; F. Renault; D. Baubichon; M. Peoc'h; M.-F. Burckhart; M. Galonnier; D. Clarençon; N. Jourdil (pp. 480-486).

We performed an experiment to characterize the toxicity of soman in cynomolgus monkeys in which organophosphorus intoxication was followed by treatment with either the current three-drug therapy atropine/pralidoxime/diazepam or a combination of atropine/pralidoxime/avizafone, avizafone being the water soluble prodrug of diazepam. Clinical, electrophysiological, and histological approaches were combined. When benzodiazepines were injected at the similar molar dose of 0.7 µmol/kg, the protection against soman toxicity was better with the atropine/pralidoxime/diazepam combination than with the atropine/pralidoxime/avizafone one. Pharmacokinetic studies demonstrated that this difference of efficacy could be explained by a lower plasmatic load of diazepam obtained after injection of avizafone at 0.7 µmol/kg, compared to the administration of diazepam at the same molar dose. Moreover, after injection of avizafone, plasmatic levels of diazepam were achieved faster and declined more rapidly than after administration of diazepam. Compared to diazepam given at a dose of 0.7 µmol/kg, injection of 1 µmol avizafone/kg gave a similar plasmatic load of benzodiazepine, but with a lower time to maximum plasma concentration (t _max) and a higher maximum plasma concentration (C _max) for plasmatic diazepam. We therefore went on to demonstrate that administration of the atropine/pralidoxime/avizafone combination at a dose 1 µmol benzodiazepine/kg to intoxicated monkeys afforded electrophysiological and histological protection similar to that obtained after administration of atropine/pralidoxime/diazepam at a dose of 0.7 µmol diazepam/kg. Reflections on the possible incorporation of avizafone in three-drug emergency treatment are presented.

Keywords: Diazepam Avizafone Seizures Neuropathology Organophosphate poisoning

No Title by Yoshimasa Kinoshita; Hirohiko Matsumura; Hideki Igisu; Akira Yokota (pp. 487-489).

When rats were injected intraperitoneally with acrylamide (50 mg/kg per day) for 8 days, all animals developed ataxia and weakness in the hindlimbs. On examining their brain with an ultrahigh-field (4.7 T) magnetic resonance (MR) spectrometer, the lateral ventricles on both sides and the third ventricle were dilated. The aqueduct and cisterns were also enlarged. The size of the cerebral cortex was quantified in three MR image slices covering the cerebrum. Compared with the images of the brain of body weight-matched controls, the cerebral cortex of rats intoxicated with acrylamide was found to be smaller in the primary motor area in all slices, and in the primary or secondary sensory area in two slices. Taken together with previous enzymatic analyses, rats intoxicated with acrylamide (50 mg/kg per day for 8 days) seem to represent an animal model of acrylamide encephalopathy not only biochemically but also structurally.

Keywords: Acrylamide Magnetic resonance Brain Neurotoxicity

No Title by J. Bünger; J. Krahl; K. Baum; O. Schröder; M. Müller; G. Westphal; P. Ruhnau; T. G. Schulz; E. Hallier (pp. 490-498).

Diesel engine exhaust particles (DEP) contribute substantially to ambient air pollution. They cause acute and chronic adverse health effects in humans. Biodiesel (rapeseed oil methyl ester, RME) is used as a "green fuel" in several countries. For a preliminary assessment of environmental and health effects of RME, the particulate-associated emissions from the DEP of RME and common fossil diesel fuel (DF) and their in vitro cytotoxic and mutagenic effects were compared. A test tractor was fuelled with RME and DF and driven in a European standard test cycle (ECE R49) on an engine dynamometer. Particle numbers and size distributions of the exhausts were determined at the load modes "idling" and "rated power". Filter-sampled particles were extracted and their cytotoxic properties tested using the neutral red assay. Mutagenicity was tested using the Salmonella typhimurium/microsome assay. Despite higher total particle emissions, solid particulate matter (soot) in the emissions from RME was lower than in the emissions from DF. While the size distributions and the numbers of emitted particles at "rated power" were nearly identical for the two fuels, at "idling" DF emitted substantially higher numbers of smaller particles than RME. The RME extracts caused fourfold stronger toxic effects on mouse fibroblasts at "idling" but not at "rated power" than DF extracts. The extracts at both load modes were significantly mutagenic in TA98 and TA100. However, extracts of DF showed a fourfold higher mutagenic effect in TA98 (and twofold in TA100) than extracts of RME. These results indicate benefits as well as disadvantages for humans and the environment from the use of RME as a fuel for tractors. The lower mutagenic potency of DEP from RME compared to DEP from DF is probably due to lower emissions of polycyclic aromatic compounds. The higher toxicity is probably caused by carbonyl compounds and unburned fuel, and reduces the benefits of the lower emissions of solid particulate matter and mutagens from RME.

Keywords: Biodiesel Rapeseed oil Diesel engine emissions Particulate matter Ultrafine particles Polycyclic aromatic hydrocarbons Mutagenicity Cytotoxicity

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Archives of Toxicology (v.74, #8)