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Archives of Toxicology (v.81, #4)

The enhancement of the subacute repeat dose toxicity test OECD TG 407 for the detection of endocrine active chemicals: comparison with toxicity tests of longer duration by Heinz-Peter Gelbke; Andreas Hofmann; J. William Owens; Alexius Freyberger (pp. 227-250).

The “enhanced TG 407” will reliably identify chemicals with a strong to moderate potential to act through endocrine modes of action on the gonads and the thyroid. In addition, this test method gives a first indication for the dose-related potency.Substances with a low potency for an endocrine mode of action, i.e., having only marginal effects in the most comprehensive in vivo studies such as multi-generation studies, may not elicit clear endocrine-related effects in the “enhanced TG 407”. In these cases, the primary or principal effects observed will be driven by other toxic actions of the test materials in the “enhanced TG 407”.It may be concluded from the present database that prolongation of exposure from 28 days up to 90 days is unlikely to improve the chance of detecting an endocrine-mediated effectA number of higher tier studies with in utero and pubertal exposure show that prenatally exposed rats may be more sensitive to exposures to compounds with very low estrogenic or antiandrogenic potential in some cases than young adult rats as used in the “enhanced TG 407”.Overall, these comparisons support the use of the “enhanced TG407” for the detection of endocrine active chemicals. It is therefore recommended to fully accept the enhancements and include them in the test method for toxicological and regulatory use.

Keywords: Repeat dose toxicity tests; OECD 28 days test guideline 407; (anti)estrogens, (anti)androgens, thyroid active substances; Comparison of dose response relationships

Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells by Patricia Conde; Leonor C. Acosta-Saavedra; Raquel C. Goytia-Acevedo; Emma S. Calderon-Aranda (pp. 251-259).

A proposed mechanism for the As-induced inhibition of cell proliferation is the inhibition of IL-2 secretion. However, the effects of arsenite on IL-2 mRNA expression or on the ERK pathway in activated-T cells have not yet been described. We examined the effect of arsenite on IL-2 mRNA expression, cell activation and proliferation in PHA-stimulated murine lymphocytes. Arsenite (1 and 10 μM) decreased IL-2 mRNA expression, IL-2 secretion and cell proliferation. Arsenite (10 μM) strongly inhibited ERK-phosphorylation. However, the partial inhibition (50%) of IL-2 mRNA produced by 1 μM, consistent with the effects on IL-2 secretion and cell proliferation, could not be explained by the inhibition of ERK-phosphorylation, which was not affected at this concentration. The inhibition of IL-2 mRNA expression caused by 1 μM could be associated to effects on pathways located downstream or parallel to ERK. Arsenite also decreased early activation (surface CD69⁺ expression) in both CD4⁺ and CD8⁺, and decreased total CD8⁺ count without significantly affecting CD4⁺, supporting that the cellular immune response mediated by cytotoxic T cells is an arsenic target. Thus, our results suggest that arsenite decreases IL-2 mRNA levels and T-cell activation and proliferation. However, further studies on the effects of arsenite on IL-2 gene transcription and IL-2 mRNA stability are needed.

Keywords: Sodium arsenite; IL-2 mRNA; T-cell activation; Cell proliferation; Phosphorylated ERKs

Internal dose-effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in gonadotropin-primed weanling rat model by Mariko Shirota; Toyozo Kaneko; Mitsunobu Okuyama; Yosuke Sakurada; Kinji Shirota; Yasuhiko Matsuki (pp. 261-269).

Single sc injection of 5 IU equine chorionic gonadotropin (eCG) induces ovulation in weanling female rats 3 days later. It has been shown that treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24 h before eCG injection reduces eCG-stimulated ovarian hypertrophy and inhibits ovulation. The present study intended to compare internal dose-effects of TCDD between these endpoints and representative endpoints for TCDD toxicity, such as weights of the liver and thymus, in weanling female rats given orally 0, 1, 4 or 16 μg/kg TCDD 24 h before eCG injection on postnatal day 25. Measurement of plasma TCDD concentrations by ELISA at 6, 72 and 96 h after TCDD revealed that significant levels of TCDD were maintained in systemic circulation until 96 h (on the day of induced ovulation) with the highest level at 6 h after TCDD treatment. Ovarian TCDD concentrations varied similarly and tended to be higher than those in the thymus at all time points, whereas hepatic concentrations of TCDD were the highest among the tissues. Although ≥ 4 μg/kg TCDD affected the weights of the thymus and liver, no differences were observed in ovarian weights at any time point or in ovulation between corn oil-treated and TCDD-treated groups. Furthermore, ovarian levels of representative mRNAs in follicles were not affected by TCDD treatment. Since TCDD increased the amount of cytochrome P450 1A1 mRNA in the ovary, the administered TCDD stimulated the aryl hydrocarbon receptor-signaling pathway. From these results, we concluded that thymus weights of weanling female rats responded to TCDD at a lower internal dose as compared with that ovarian hypertrophy and follicular growth from early antral stage to ovulation would respond to.

Keywords: TCDD; Weanling rat; Ovary; Internal dose-effects; Thymus; CYP1A1; Equine chorionic gonadotrophin; ELISA

Pattern of MAP kinases p44/42 and JNK activation by non-lethal doses of tributyltin in human natural killer cells by Aloice O. Aluoch; Sabah O. Odman-Ghazi; Margaret M. Whalen (pp. 271-277).

Tributyltin (TBT) has been shown to disrupt the ability of natural killer (NK) cells to destroy tumor targets in vitro even at exposures of 25 nM for 24 h, but cell viability was not significantly impacted. Thus, evaluation of intracellular molecular events that regulate cell viability in TBT exposed NK cells are of interest. It has been suggested that activation of the mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), may promote apoptosis while activation of the MAPK p44/42 may be crucial in mediating anti-apoptotic stimuli. However, it is well established that increases in pro-apoptotic BCL-2 family members, such as Bax, results in cell death. We have set out to study the effects of a range of TBT concentrations on the MAPKs, JNK and p44/42. Additionally, we examined the effects of TBT on the levels of pro-apoptotic proteins Bax and p53 as well as anti-apoptotic protein Bcl-2. The results show that 300–25 nM TBT activated JNK within 10 min. MAPK p44/42 was also activated by 300–50 nM TBT within 10 min. These data show that while 300–200 nM TBT activates p44/42 significantly more than JNK, the pattern of 100–25 nM TBT activation of these MAPKs may be similar. TBT exposure alters neither pro-apoptotic proteins Bax and p53 nor anti-apoptotic protein Bcl-2 levels at any exposure studied. The results suggest that exposure to TBT activated the anti-apoptotic regulatory p44/42 pathway to a greater extent than the pro-apoptotic JNK pathway, which may explain to some extent how NK cell viability is maintained.

Apoptosis induced by acrylamide in SH-SY5Y cells by Tomoyuki Sumizawa; Hideki Igisu (pp. 279-282).

Acrylamide (1–5 mM) dose-dependently decreased cell viability in human neuroblastoma cells (SH-SY5Y). The caspase-3 activity and cell population in sub-G₁ phase were elevated and peaked on exposure to 3 mM acrylamide, while both were less so at higher dose (4 and 5 mM). Z-VAD-fmk, a pan-caspase inhibitor, lowered the apparent cytotoxicity of acrylamide. U0126, a specific inhibitor of extracellular signal-regulated protein kinase (ERK) kinase, suppressed the elevation of caspase-3 activities as well as that of sub-G₁ population. Thus, although mechanisms other than caspase-dependent apoptosis may be involved, apoptotic process seems to take place in the genesis of toxicity of acrylamide in SH-SY5Y cells through ERK pathway and activation of caspase-3.

Keywords: Acrylamide; SH-SY5Y cells; Caspase-3; Apoptosis; ERK

Effects of combined, multiple stressors on pyridostigmine-induced acute toxicity in rats by Praveena Baireddy; Nikita Mirajkar; Anuradha Nallapaneni; Nicole Singleton; Carey N. Pope (pp. 283-289).

A number of studies have evaluated the possibility that stress-induced changes in blood–brain barrier permeability enhanced the central effects of the carbamate acetylcholinesterase inhibitor, pyridostigmine. We previously found relatively little evidence of stress-induced changes in the acute toxicity of pyridostigmine in rats using a variety of restraint, forced running and forced swimming stress conditions. In this study, we evaluated the effects of sequential pre-exposure to multiple stressors on the acute toxicity of pyridostigmine. Rats (n = 8 per treatment group) were either un-stressed or stressed by restraint (60 min), forced running (60 min, 15 m/min, 6° incline) and forced swimming (15 min), and then given either vehicle (saline, 1 ml/kg, po) or pyridostigmine (30 mg/kg, po) immediately after the final stressor. Functional signs of cholinergic toxicity (involuntary movements, autonomic dysfunction) were recorded at 0.5, 1 and 2 h after dosing. Body temperature was measured both before stress and 2 h after dosing. Rats were sacrificed immediately after 2-h functional observations to collect tissues (whole blood, diaphragm, frontal cortex, hippocampus and cerebellum) for measurement of cholinesterase activity. Stressed rats treated with pyridostigmine exhibited higher lethality (2/8) compared to unstressed rats given pyridostigmine (0/8). Pyridostigmine elicited classical signs of cholinergic toxicity, but the rats that died did not show increased cholinergic signs and no significant differences in cholinergic signs were noted between treatment groups. Cholinesterase activity was significantly inhibited in blood (47–50%) and diaphragm (80%) following pyridostigmine exposure regardless of stress conditions. Slight but significant inhibition (11–15%) of cerebellar cholinesterase activity was observed following pyridostigmine exposure, but inhibition was not influenced by stress. We conclude that while acute lethality from pyridostigmine may be increased by combined, multiple stressors, increased lethality does not appear due to enhanced cholinergic toxicity or via increased cholinesterase inhibition in either central or peripheral tissues.

Keywords: Cholinesterase inhibition; Blood–brain barrier; Restraint; Treadmill; Forced swimming; Functional toxicity

Role of bile in pathogenesis of indomethacin-induced enteropathy by Molly Jacob; Russel Foster; Gudmundur Sigthorsson; Robert Simpson; Ingvar Bjarnason (pp. 291-298).

Ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) causes an enteropathy. The pathogenesis involves biochemical initiation of intestinal mucosal damage due to NSAID-induced inhibition of cyclooxygenase and the topical effects of these drugs. These effects lead to increased intestinal permeability and inflammation. Luminal bile acids play a controversial role in the damage produced by these drugs. The aim of this study was to determine the role of bile in producing the enteropathy caused by indomethacin, an NSAID commonly used in toxicity studies. Sprague–Dawley rats were subjected to bile duct ligation. Twenty-four hours later, they were dosed with indomethacin. Intestinal permeability (⁵¹Cr-EDTA) and inflammation (faecal calprotectin) were measured in the animals at various time periods after the dose. Intestinal permeability was significantly higher in rats 1–6 h after dosing with indomethacin, but not at 24–29 h or day 4, when compared with corresponding values for control animals. Excretion of faecal calprotectin was elevated in the indomethacin-treated rats. The drug-treated animals showed no evidence of ulceration when they were sacrificed 29 h or a week after the dose of indomethacin. Bile acids per se did not affect intestinal permeability or faecal excretion of calprotectin, when given along with indomethacin or its vehicle. We conclude that macroscopic small bowel damage does not occur with indomethacin if bile is excluded, despite the induction of permeability and inflammation. This study highlights the importance of luminal factors, such as bile, in producing indomethacin-induced ulceration in the rat small intestine.

Keywords: Anti-inflammatory drugs (non-steroidal); Bile; Enteropathy; Indomethacin; Inflammation; Small intestinal disease

Inhalation of diluted diesel engine emission impacts heart rate variability and arrhythmia occurrence in a rat model of chronic ischemic heart failure by Frédéric Anselme; Stéphane Loriot; Jean-Paul Henry; Frédéric Dionnet; Jean-Gérard Napoleoni; Christian Thuillez; Jean-Paul Morin (pp. 299-307).

Both increase in cardiac arrhythmia incidence and decrease in heart rate variability (HRV) have been described following human and experimental animal exposures to air pollutants. However, the potential causal relationship between these two factors remains unclear. Incidence of ventricular arrhythmia and HRV were evaluated during and after a 3 h period of Diesel engine exhaust exposure in ten healthy and ten chronic ischemic heart failure (CHF, 3 months after coronary ligation) Wistar rats using implantable ECG telemetry. Air pollutants were delivered to specifically designed whole body individual exposure chambers at particulate matter concentrations similar to those measured inside cabins of cars inserted in congested urban traffic. Recordings were obtained from unrestraint and unsedated vigil rats. Immediate decrease in RMSSD was observed in both healthy (6.64 ± 2.62 vs. 4.89 ± 1.67 ms, P < 0.05) and CHF rats (8.01 ± 0.89 vs. 6.6 ± 1.37 ms, P < 0.05) following exposure. An immediate 200–500% increase in ventricular premature beats was observed in CHF rats only. Whereas HRV progressively returned to baseline values within 2.5 h after exposure start, the proarrhythmic effect persisted as late as 5 h after exposure termination in CHF rats. Persistence of ventricular proarrhythmic effects after HRV normalization suggests that HRV reduction is not the mechanism of cardiac arrhythmias in this model. Our methodological approach, closely reflecting the real clinical situations, appeared to be a unique tool to provide further insight into the pathophysiological mechanisms of traffic related airborne pollution health impact.

Keywords: Chronic heart failure; Heart rate variability; Arrhythmia; ECG telemetry; Diesel engine emissions; Inhalation toxicology

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Archives of Toxicology (v.81, #4)