Toxicological Sciences - Advance Access

Acute treatment with diphenyl diselenide inhibits glutamate uptake into rat hippocampal slices and modifies glutamate transporters, SNAP-25 and GFAP immunocontent

Wed, 18 Nov 2009 23:21:32 PST

Diphenyl diselenide (PhSe)₂ is a selenium organic compound that has been described to inhibit glutamate binding at synaptic membranes and uptake into cortical slices, but there are no studies about its effects on glutamate transporters and related synaptic proteins. Hippocampal slices from rats treated acutely with (PhSe)₂ (1, 10 and 100 mg/kg, oral route) were evaluated on glutamate uptake, redox state, the immunocontent of glial (GLAST and GLT1), neuronal (EAAC1) and vesicular VGLUT1 glutamate transporters. Besides, cell viability was evaluated by glial fibrilar acid protein (GFAP) and synaptosomal-associated protein 25 (SNAP-25) immunocontent and DAPI and Fluoro Jade C staining. Hippocampal slices from rats treated with (PhSe)₂ exhibited a non-dose dependent inhibition of glutamate uptake (53, 38 and 45 %, respectively). All doses increased EAAC1, decreased SNAP-25, did not modify GLT1 immunocontent and there was no evidence of oxidative stress. (PhSe)₂ (100 mg/kg) increased 32 % GLAST, decreased 34 % VGLUT1 and 21 % GFAP immunocontent. Besides, (PhSe)₂ (100 mg/kg) decreased by 25 % GFAP-stained astrocytes and 27 % DAPI-stained cells in the CA1 subfield. Our results suggest that the increase of EAAC1 and GLAST immunocontent by (PhSe)₂ might be a compensatory mechanism by surviving cells in order to reduce extracellular glutamate levels, avoiding possible neurotoxic effects. The impairment of glutamate uptake by the highest dose of (PhSe)₂ seems to be related to a decrease on VGLUT1, SNAP-25 and damage to astrocytes. Since there were no signs of oxidative stress, our findings revealed that depending on the dose acute administration of (PhSe)₂ causes modifications in important synaptic-related proteins and damage to the astrocytes and these events must be taken into account in its pharmacological properties.

Pharmacokinetics of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCPy) in rat saliva

Smith, J. N., Wang, J., Lin, Y., Timchalk, C. — Tue, 17 Nov 2009 06:22:06 PST

Biological monitoring (biomonitoring) to quantify systemic exposure to the organophosphorus insecticide chlorpyrifos (CPF) has historically focused on the quantitation of major CPF metabolites in urine. Non-invasive techniques are being advocated as novel means of biomonitoring for a variety of potential toxicants, including pesticides (like CPF); and saliva has been suggested as an ideal body fluid. However in order to be acceptable, there is a need to understand salivary pharmacokinetics of CPF metabolites in order to extrapolate saliva measurements to whole body exposures. In this context, in vivo pharmacokinetics of 3,5,6-trichloro-2-pyridinol (TCPy), the major chemical specific metabolite of CPF, was quantitatively evaluated in rat saliva. Experimental results suggest that TCPy partitioning from plasma to saliva in rats is relatively constant over a range of varying physiological conditions. TCPy pharmacokinetics were very similar in blood and saliva (area under the curve (AUC) values were proportional and elimination rates ranged from 0.007 to 0.019 h^-1) and saliva/blood TCPy concentration ratios were not affected by TCPy concentration in blood (p = 0.35) or saliva flow rate (p = 0.26). The TCPy concentration in saliva was highly correlated to the amount of unbound TCPy in plasma (r = 0.96), and the amount TCPy protein-binding in plasma was substantial (98.5%). The median saliva/blood concentration ratio (0.049) was integrated as a saliva/blood TCPy partitioning coefficient within an existing physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for CPF. The model was capable of accurately predicting TCPy concentrations in saliva over a range of blood concentrations. These studies suggest that saliva TCPy concentration can be utilized to ascertain CPF exposure. It is envisioned that the PBPK/PD can likewise be used to estimate CPF dosimetry based on the quantitation of TCPy in spot saliva samples obtained from biomonitoring studies.

Liver Specific Ablation of Integrin Linked Kinase (ILK) in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration

Tue, 17 Nov 2009 06:22:06 PST

We have recently demonstrated that disruption of ECM/Integrin signaling via elimination of Integrin linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by Phenobarbital (PB). WT (wild type) and ILK/Liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5 and 10 days during PB administration. In the hepatocyte-specific ILK/Liver-/- mice the liver/body weight ratio was more than double as compared to 0h at day 2 (2.5 times) while at day 5 and 10 it was enlarged three times. In the WT mice the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased PCNA positive cells in the ILK/Liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals the proliferative response had come back to normal by day 5 and 10. Hepatocytes of the ILK/Liver-/- mice continued to proliferate up until day 10. ILK/Liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens such as phenobarbital.

Evaluation of human interindividual variation in bioactivation of estragole using physiologically based biokinetic (PBBK) modeling

Tue, 17 Nov 2009 06:22:05 PST

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1'-hydroxyestragole, due to variation in two key metabolic reactions involved in formation and detoxification of this metabolite, namely 1'-hydroxylation of estragole and oxidation of 1'-hydroxyestragole. Formation of 1'-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1 and results of the present study support that oxidation of 1'-hydroxyestragole is catalyzed by 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2). In a first approach, the study defines physiologically-based biokinetic (PBBK) models for fourteen individual human subjects, revealing a 1.8-fold interindividual variation in the area under the concentration-time curve (AUC) for 1'-hydroxyestragole in the liver within this group of human subjects. Variation in oxidation of 1'-hydroxyestragole by 17β-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1'-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99^th percentile divided by the 50^th percentile of the predicted distribution of the AUC of 1'-hydroxyestragole in the liver. The CSAF was estimated to range between 1.7-4.0, depending on the level of variation that was taken into account for oxidation of 1'-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the population.

Triclosan and Anuran Metamorphosis: No Effect on Thyroid-Mediated Metamorphosis in Xenopus laevis

Mon, 16 Nov 2009 06:34:25 PST

Nieuwkoop and Faber (NF) stage 51 Xenopus laevis larvae were exposed for 21 d to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 µg TCS/L. Primary endpoints were survival, hind limb length, body length (whole; snout-to-vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure d 21 specimens. TH receptor-beta (TRβ) expression was measured in stage-matched tail fin tissue samples collected at exposure d 0 and 21. Reduced larval growth occurred at exposure d 21 with 1.5 µg/L treatment. Larval developmental stage at exposure d 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxin (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRβ expression in exposure d 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5- or 7.2-µg TCS/L. Our study indicates that environmentally-relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.

Roles of rat renal organic anion transporters in transporting perfluorinated carboxylates with different chain lengths

Weaver, Y. M., Ehresman, D. J., Butenhoff, J. L., Hagenbuch, B. — Fri, 13 Nov 2009 09:28:47 PST

Perfluorinated carboxylates (PFCAs) are generally stable to metabolic and environmental degradation and have been found at low concentrations in environmental and biological samples. Renal clearance of PFCAs depends on chain length, species, and, in some cases, gender within species. While perfluoroheptanoate (C7) is almost completely eliminated renally in both male and female rats, renal clearance of perfluorooctanoate (C8) and perfluorononanoate (C9) is much higher in female rats. Perfluorodecanoate (C10) mainly accumulates in the liver for both genders. Therefore, we tested whether PFCAs with different chain lengths are substrates of rat renal transporters with gender specific expression patterns. Inhibition of uptake of model substrates was measured for the basolateral Oat1 and Oat3, and the apical Oat2, Oatp1a1 and Urat1 with 10µM PFCAs with chain lengths from 2-18 (C2 – C18) carbons. Perfluorohexanoate (C6), C7, and C8 inhibited Oat1-mediated p-aminohippurate transport, with C7 being the strongest inhibitor. C8 and C9 were the strongest inhibitors for Oat3-mediated estron-3-sulfate transport, while Oatp1a1-mediated estradiol-17β-glucuronide uptake was inhibited by C9, C10, and perflouroundecanoate (C11), with C10 giving the strongest inhibition. No strong inhibitors were found for Oat2 or Urat1. Kinetic analysis was performed for the strongest inhibitors. Oat1 transported C7 and C8 with K_m values of 50.5 µM and 43.2 µM, respectively. Oat3 transported C8 and C9 with K_m values of 65.7 µM and 174.5 µM, respectively. Oatp1a1 mediated transport yielded K_m values of 126.4 µM (C8), 20.5 µM (C9), and 28.5 µM (C10). These results suggest that Oat1 and Oat3 are involved in renal secretion of C7 to C9, while Oatp1a1 can contribute to the reabsorption of C8 through C10, with highest affinities for C9 and C10.

Experimental exposure to 1,1,1,3,3-Pentafluoropropane (HFC-245fa): Uptake and disposition in humans

Ernstgard, L., Andersen, M., Dekant, W., Sjogren, B., Johanson, G. — Fri, 13 Nov 2009 09:28:47 PST

The aim of this study was to determine the toxicokinetics of inhaled 1,1,1,3,3-pentafluoropropane (HFC-245fa) in humans. Five healthy volunteers of each sex were exposed in random order to 0, 100 or 300 ppm HFC-245fa for 2 h at light exercise (50W) in an exposure chamber. Capillary blood, urine and exhaled air were sampled up to 22 h post-exposure and analyzed for HFC-245fa. In addition, the metabolites fluoride, 3,3,3-trifluoropropionic acid (TFPA) and trifluoroacetic acid (TFAA) were analyzed in urine. Symptoms of irritation and central nervous system effects were rated in Visual Analogue Scales. Various biochemical (ASAT, ALAT, ALP, GGT, urate, CK, CK-MB) and inflammatory markers (SAA protein, fibrinogen, D-dimer, C-reactive protein, IL-6) in plasma were analyzed. The initial increase in blood was fast and an apparent steady-state was reached within a few minutes at both exposure levels. The post-exposure decrease in blood was equally fast and parallel to that in exhaled air. Only minor amounts of unchanged HFC-245fa were excreted in breath (0.7% of inhaled) and urine (0.001%). The observed time courses in blood and breath agreed reasonably well those obtained by physiologically based pharmacokinetic (PBPK) modelling. The PBPK simulations indicate a relative uptake during exposure of 2.1%. TFPA was not detected in urine and no increase in TFAA or fluoride above background was seen, suggesting little or no metabolism, the calculated minimum detectable metabolism being 0.001% of the inhaled amount. The symptom ratings revealed no HFC-245fa-related effects. None of the biochemical markers was affected. The changes in inflammatory markers, some of which statistically significant, were not consistent with an inflammatory response.

The Absence of the Pro-Antioxidant Transcription Factor Nrf2 Exacerbates Experimental Autoimmune Encephalomyelitis

Johnson, D. A., Amirahmadi, S., Ward, C., Fabry, Z., Johnson, J. A. — Thu, 12 Nov 2009 03:09:46 PST

Multiple sclerosis (MS) is an autoimmune disease characterized by peripheral activation of CD4⁺ T cells that migrate into the central nervous system (CNS) and mount an autoimmune neuroinflammatory attack on myelin and oligodendrocytes. Secondary to these events, however equally destructive, is the generation of inflammatory-mediated reactive oxygen and nitrogen species secreted by persistently activated microglia and astrocytes. Nuclear factor-erythroid 2–related factor 2 (Nrf2) is a basic leucine zipper transcription factor that regulates genetic expression of many protective antioxidant and detoxication enzymes. Here we describe the Nrf2-modulation of innate and adaptive immune responses in an acute autoimmune model of multiple sclerosis, EAE. Wild type (WT) mice and Nrf2-knock out (Nrf2-KO) mice were immunized with myelin oligodendrocyte glycoprotein (MOG 35-55) and monitored daily for clinical scores of disease. Disruption of Nrf2 resulted in a more severe clinical course, a more rapid onset, and a greater percentage of mice with the disease. Furthermore, increased immune cell infiltration and glial cell activation in spine was observed. In conjunction, we observed increased inflammatory enzyme (iNOS, phox-47, gp91-phox and phox-67), cytokine (IFN-gamma, IL1-b, TNF-alpha, and IL-12) and chemokine (BLC, MIG) gene expression levels in the Nrf2 deficient mice compared to the WT mice, supporting the notion that Nrf2 can modulate an autoimmune neuroinflammatory response. Our results show that the absence of Nrf2 exacerbates the development of EAE and thus suggests that activation of Nrf2 may then attenuate pathogenesis of autoimmune diseases such as multiple sclerosis as well as neurodegenerative diseases.

Short-term Exposure to Triclosan Decreases Thyroxine In Vivo via Upregulation of Hepatic Catabolism in Young Long-Evans Rats

Paul, K. B., Hedge, J. M., DeVito, M. J., Crofton, K. M. — Thu, 12 Nov 2009 03:09:45 PST

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a chlorinated phenolic antibacterial compound found in consumer products. In vitro human pregnane X receptor (PXR) activation, hepatic Phase I enzyme induction, and decreased in vivo total thyroxine (T4) suggest adverse effects on thyroid hormone homeostasis. Current research tested the hypothesis that triclosan decreases circulating T4 via upregulation of hepatic catabolism and transport. Weanling female Long-Evans rats received triclosan (0-1000 mg/kg/day) by gavage for four days. Whole blood and liver were collected 24 hrs later. Total serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay. Hepatic microsomal assays measured ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-deethylase (PROD), and uridine diphosphate glucuronyltransferase (UGT) enzyme activities. The mRNA expression of cytochrome P450s 1a1, 2b1/2, and 3a1/23, UGTs 1a1, 1a6, and 2b5, sulfotransferases 1c1 and 1b1, and hepatic transporters Oatp1a1, Oatp1a4, Mrp2, and Mdr1b was measured by quantitative RT-PCR. Total T4 decreased dose-responsively, down to 43% of control at 1000 mg/kg/day. Total T3 was decreased to 89% and 75% of control at 300 and 1000 mg/kg/day. TSH did not change. Triclosan dose-dependently increased PROD activity up to 900 percent of control at 1000 mg/kg/day. T4-glucuronidation increased nearly 2-fold at 1000 mg/kg/day. Cyp2b1/2 and Cyp3a1/23 mRNA expression levels were induced 2-fold and 4-fold at 300 mg/kg/day. Ugt1a1 and Sult1c1 mRNA expression increased 2.2-fold and 2.6-fold at 300 mg/kg/day. Transporter mRNA expression levels were unchanged. These data denote important key events in the mode-of-action for triclosan-induced hypothyroxinemia in rats, and suggest that this effect may be partially due to upregulation of hepatic catabolism, but not due to mRNA expression changes in the tested hepatic transporters.

Altered Cardiac Function and Ventricular Septal Defect in Avian Embryos Exposed to Low-Dose Trichloroethylene

Thu, 12 Nov 2009 03:09:45 PST

Trichloroethylene (TCE) is the most frequently reported organic groundwater contaminant in the U.S. It is controversial whether gestational TCE exposure causes congenital heart defects. The basis for TCE's proposed cardiac teratogenicity is not well understood. We previously showed that chick embryos exposed to 8 ppb TCE during cardiac morphogenesis have reduced cardiac output and increased mortality. To further investigate TCE's cardioteratogenic potential, we exposed in ovo chicken embryos to TCE and evaluated the heart thereafter. Significant mortality was observed following TCE exposures of 8-400 ppb during a narrow developmental period (Hamburger-Hamilton [HH] stages 15-20, embryo day ED2.3-3.5) that is characterized by myocardial expansion, secondary heart looping, and endocardial cushion formation. Of the embryos that died, most did so between ED5.5 and ED6.5. Echocardiography of embryos at ED5.5 found that TCE-exposed hearts displayed significant functional and morphological heterogeneity affecting heart rate, left ventricular mass and wall thickness. Individual embryos were identified with cardiac hypertrophy as well as hypoplasia. Chick embryos exposed to 8 ppb TCE at HH17 that survived to hatch exhibited a high incidence (38%; p < 0.01; n=16) of muscular ventricular septal defects (VSDs) as detected by echocardiography and confirmed by gross dissection; no VSDs were found in controls (n=14). The TCE-induced VSDs may be secondary to functional impairments that alter cardiac hemodynamics and subsequent ventricular foramen closure, an interpretation consistent with recent demonstrations that TCE impairs calcium handling in cardiomyocytes. These data demonstrate that TCE is a cardiac teratogen for chick.

The biological mechanisms and physicochemical characteristics responsible for driving fullerene toxicity

Johnston, H. J., Hutchison, G. R., Christensen, F. M., Aschberger, K., Stone, V. — Mon, 09 Nov 2009 06:07:34 PST

This review provides a comprehensive, critical review of the available literature purporting to assess the toxicity of carbon fullerenes. This is required as prior to the widespread utilisation and production of fullerenes, it is necessary to consider the implications of exposure for human health. Traditionally, fullerenes are formed from 60 carbon atoms, arranged in a spherical, cage-like structure. However, manipulation of surface chemistry, and molecular make-up has created a diverse population of fullerenes, which exhibit drastically different behaviour. The cellular processes that underlie observed fullerene toxicity will be discussed, and includes oxidative, genotoxic, and cytotoxic responses. The antioxidant/cytoprotective properties of fullerenes (and the attributes responsible for driving these phenomena) have been considered, and encourages their utilisation within the treatment of oxidant mediated disease. A number of studies have focussed on improving the water solubility of fullerenes, in order to enable their exploitation within biological systems. Manipulating fullerene water solubility has included the use of surface modifications, solvents, extended stirring, and mechanical processes. However, the ability of these processes to also impact on fullerene toxicity requires assessment, especially when considering the use of solvents, which particularly appear to enhance fullerene toxicity. A number of the discussed investigations were not conducted to reveal if fullerene behaviour was due to their nanoparticle dimensions, but instead addressed the biocompatibility and toxicity of fullerenes. The hazards to human health, associated with fullerene exposure, are uncertain at this time, and further investigations are required to decipher such effects before an effective risk assessment can be conducted.

Cadmium Induces Intracellular Ca2+- and H2O2-dependent Apoptosis through JNK- and p53-mediated Pathways in Skin Epidermal Cell line

Son, Y.-O., Lee, J.-C., Hitron, J. A., Pan, J., Zhang, Z., Shi, X. — Tue, 03 Nov 2009 07:18:39 PST

Cadmium is a toxic heavy metal and has been widely used in industry. The skin is an important target for this metal. The mechanisms by which cadmium leads to damage to the skin are unclear at present. The aims of this study were to examine whether cadmium induces apoptosis in mouse skin epidermal cell line, JB6 Cl41 cells, and to investigate the cellular mechanisms by which cadmium causes cytotoxicity in the cells. The present study showed that cadmium induced cell death by apoptosis in a dose-dependent manner, as proven by the appearance of cell shrinkage, the increase of Annexin V-positively stained cells, and the formation of nuclear DNA ladders. Cadmium-induced apoptosis involved a mitochondria-mediated mechanism but not caspase-dependent pathway, in that the critical apoptotic events induced by cadmium, such as the decrease of Bcl-2/Bcl-xL, the increase of GADD45, and the nuclear translocation of AIF, were not affected by the inhibition of executive caspases. In contrast, blockage of p53 and JNK by pharmacological inhibitors or si-RNA transfection suppressed the cadmium-induced apoptosis with the concomitant inhibition of anti-apoptotic Bcl-2 family proteins and GADD45, respectively. Further, the activation of p53 and JNK and their downstream proteins in cadmium-exposed cells were inhibited by individual treatment with catalase and Bapta-AM. These results suggest that cadmium induces apoptosis via the activation of JNK- and p53-mediated signaling, where calcium ion and hydrogen peroxide act as the pivotal mediators of the apoptotic signaling.

Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species

Sun, 01 Nov 2009 23:06:30 PST

Relative potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were determined in vitro in primary hepatocyte cultures of chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and Japanese quail (Corturnix japonica) embryos. Concentration-dependent effects on ethoxyresorufin O-deethylase (EROD) activity, and expression of cytochrome P4501A4 and 1A5 (CYP1A4 and CYP1A5) mRNA were determined in hepatocytes exposed to serial dilutions of TCDD, PeCDF or TCDF for 24 h. In chicken hepatocytes, the three compounds were equipotent inducers of EROD activity and CYP1A4/5 mRNA expression. However, in ring-necked pheasant and Japanese quail hepatocytes, PeCDF was more potent than TCDD (3- to 5-fold in ring-necked pheasant; 13- to 30-fold in Japanese quail). Among species, the rank order of sensitivity (most to least) to EROD and CYP1A4/5 mRNA induction for TCDD and TCDF was: chicken > ring-necked pheasant > Japanese quail. In contrast, the three species were approximately equisensitive to EROD and CYP1A4/5 mRNA induction by PeCDF. It has generally been assumed that TCDD is the most potent ‘dioxin-like compound’ (DLC) and that the chicken is the most sensitive avian species to CYP1A induction by all DLCs. This study indicates that PeCDF is more potent than TCDD in ring-necked pheasant and Japanese quail hepatocytes, and that ring-necked pheasant, Japanese quail and chicken hepatocytes are equally sensitive to CYP1A induction by PeCDF.

Creation of a Hyper-permeable Yeast Strain to Genotoxic Agents through Combined Inactivation of PDR and CWP Genes

Zhang, M., Hanna, M., Li, J., Butcher, S., Dai, H., Xiao, W. — Sun, 01 Nov 2009 23:06:30 PST

We previously established a genotoxicity detection system based on the transcriptional response of the yeast RNR3 gene to DNA damage. In order to further improve its sensitivity to genotoxicants, we have attempted to increase cell permeability by removing cell wall mannoproteins (CWPs). Here, we report that selected deletion of pleiotropic drug resistance (PDR) genes encoding membrane efflux transporters also enhanced cellular sensitivity to treatment by various genotoxic agents. Furthermore, we have validated our hypothesis that PDR and CWP protect cells through different mechanisms by demonstrating that simultaneous inactivation of the above two pathways resulted in a synergistic enhancement of assay sensitivity as measured by RNR3-lacZ expression, and that this effect is at the cell permeability level. The quadruple mutation results in RNR3-lacZ assay sensitivity to tested chemicals that apparently surpasses the industry standard Ames test. We argue that this hyper-permeable yeast mutant strain would be suitable for other chemical-based genotoxic assays.

Airway Trefoil Factor Expression during Naphthalene Injury and Repair

Greeley, M. A., Van Winkle, L. S., Edwards, P. C., Plopper, C. G. — Fri, 30 Oct 2009 08:08:00 PDT

While the role of trefoil factors (TFF) in the maintenance of epithelial integrity in the gastrointestinal tract is well known, their involvement in wound healing in the conducting airway is less well understood. We defined the pattern of expression of TFF1, TFF2 and TFF3 in the airways of mice during repair of both severe (300mg/kg) and moderate (200mg/kg) naphthalene induced Clara cell injury. Quantitative real-time PCR for TFF mRNA expression and immunohistochemistry for protein expression were applied to airway samples obtained by microdissection of airway trees or to fixed lung tissue from mice at 6 and 24 hours and 4 and 7 days after exposure to either naphthalene or an oil (vehicle) control. All three frefoil factors were expressed in normal whole lung and airways. TFF2 was the most abundant and was enriched in airways. Injury of the airway epithelium by 300 mg/kg naphthalene caused a significant induction of TFF1 gene expression at 24hrs, 4 and 7 days. In contrast TFF2 was decreased in the high dose group at 24 hrs and 4 days but returned to baseline levels by 7 days. TFF3 gene expression was not significantly changed at any timepoint. Protein localization via immunohistochemistry did not directly correlate with the gene expression measurements. TFF 1 and 3 expression was most intense in the degenerating Clara cells in the injury target zone at 6 and 24 hrs. Following the acute injury phase TFF1 and 3 were localized to the luminal apices of repairing epithelial cells and to the adjacent mesenchyme in focal regions that correlated with bifurcations and the bronchoalveolar duct junction. The temporal pattern of increases in TFF1, TFF2 and TFF3 indicate a role in cell death as well as proliferation, migration and differentiation phases of airway epithelial repair.

Quantitative Risk Analysis for N-Methyl Pyrrolidone using Physiologically Based Pharmacokinetic and Benchmark Dose Modeling

Thu, 29 Oct 2009 12:06:43 PDT

Establishing an occupational exposure limit (OEL) for N-methyl pyrrolidone (NMP) is important due to its widespread use as a solvent. Based on studies in rodents, the most sensitive toxic endpoint is a decrease in fetal/pup bodyweights observed after oral, dermal, and inhalation exposures of dams to NMP. Evidence indicates the parent compound is the causative agent. To reduce the uncertainty in rat to human extrapolations, physiologically based pharmacokinetic (PBPK) models were developed to describe the pharmacokinetics of NMP in both species. Since in utero exposures are of concern, the models considered major physiological changes occurring in the dam or mother over the course of gestation. The rat PBPK model was used to determine the relationship between NMP concentrations in maternal blood and decrements in fetal/pup body weights following exposures to NMP vapor. Body weight decrements seen after vapor exposures occurred at lower NMP blood levels than those observed after oral and dermal exposures. Benchmark dose (BMD) modeling was used to better define a point of departure (POD) for fetal/pup body weight changes based on dose-response information from two inhalation studies in rats. The POD and human PBPK model were then used to estimate the human equivalent concentrations (HEC) that could be used to derive an OEL value for NMP. The geometric mean of the PODs derived from the rat studies was estimated to be 350 mg*hr/L (expressed in terms of internal dose), a value which corresponds to a HEC of 480 ppm (occupational exposure of 8 hours/day, 5 days/week). The HEC is much higher than recently-developed internationally-recognized OELs for NMP of 10 ppm to 20 ppm, suggesting that these OELs adequately protect workers exposed to NMP vapor.

Aflatoxin B1 transfer and metabolism in human placenta

Thu, 29 Oct 2009 12:06:42 PDT

Aflatoxin B1 (AFB1), a common dietary contaminant, is a major risk factor of hepatocellular carcinoma (HCC). Early onset of HCC in some countries in Africa and South-East Asia indicates the importance of early life exposure. Placenta is the primary route for various compounds, both nutrients and toxins, from the mother to the fetal circulation. Furthermore, placenta contains enzymes for xenobiotic metabolism. AFB1, AFB1-metabolites and AFB1-albumin adducts have been detected in cord blood of babies after maternal exposure during pregnancy. However, the role that the placenta plays in the transfer and metabolism of AFB1 is not clear. In this study, placental transfer and metabolism of AFB1 were investigated in human placental perfusions and in in vitro studies. Eight human placentas were perfused with 0.5 or 5 µM AFB1 for 2-4 hours. In vitro incubations with placental microsomal and cytosolic proteins from eight additional placentas were also conducted. Our results from placental perfusions provide the first direct evidence of the actual transfer of AFB1 and its metabolism to aflatoxicol (AFL) by human placenta. In vitro incubations with placental cytosolic fraction confirmed the capacity of human placenta to form AFL. AFL was the only metabolite detected in both perfusions and in vitro incubations. Since AFL is less mutagenic, but putatively as carcinogenic as AFB1, the formation of AFL may not protect the fetus from the toxicity of AFB1.

Airway exposure to silica coated TiO2 nanoparticles induces pulmonary neutrophilia in mice

Thu, 29 Oct 2009 12:06:42 PDT

The importance of nanotechnologies and engineered nanoparticles has grown rapidly. It is therefore crucial to acquire up-to-date knowledge of the possible harmful health effects of these materials. Since a multitude of different types of nanosized TiO₂ particles are used in industry we explored their inflammatory potential using mouse and cell models. BALB/c mice were exposed by inhalation for either 2 hours, 2 hours on four consecutive days or 2 hours on four consecutive days for four weeks to several commercial TiO₂ nanoparticles, SiO₂ nanoparticles and to nanosized TiO₂ generated in a gas-to-particle conversion process at 10 mg/m³. In addition, effects of in vitro exposure of human macrophages and fibroblasts (MRC-9) to the different particles were assessed. SiO₂-coated rutile TiO₂ nanoparticles (cnTiO₂) was the only sample tested that elicited clear-cut pulmonary neutrophilia. Uncoated rutile and anatase as well as nanosize SiO₂ did not induce significant inflammation. Pulmonary neutrophilia was accompanied by increased expression of TNF- and neutrophil attracting chemokine CXCL1 in the lung tissue. TiO₂ particles accumulated almost exclusively in the alveolar macrophages. In vitro exposure of murine and human macrophages to cnTiO₂ elicited significant induction of TNF- and neutrophil attracting chemokines. Stimulation of human fibroblasts with cnTiO₂-activated macrophage supernatant induced high expression of neutrophil attracting chemokines, CXCL1 and CXCL8. Interestingly, the level of lung inflammation could not be explained by the surface area of the particles, their primary or agglomerate particle size or radical formation capacity, but is rather explained by the surface coating. Our findings emphasize that it is vitally important to take into account in the risk assessment that alterations of nanoparticles, e.g. by surface coating, may drastically change their toxicological potential.

In Utero and Lactational Exposure to Bisphenol A, in contrast to Ethinyl Estradiol, Does not Alter Sexually Dimorphic Behavior, Puberty, Fertility and Anatomy of Female LE Rats

Ryan, B. C., Hotchkiss, A. K., Crofton, K. M., Gray, L. E. — Wed, 28 Oct 2009 07:41:33 PDT

Many chemicals released into the environment display estrogenic activity including the oral contraceptive ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). EE2 is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and CNS as an area of "some concern", whereas most effects were rated as of "negligible" or "minimal" concern. However, the number of robust studies in this area was limited.

The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female LE rat offspring.

Pregnant rats were gavaged with vehicle, EE2 (0.05 to 50 µg/kg/day) or BPA (2, 20 and 200 µg/kg/day) from day 7 of gestation to postnatal day 18 and the female offspring were studied. EE2 (50 µg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes and induced malformations of the external genitalia (5 µg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 µg/kg) and absence of lordosis behavior (15 µg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically-relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat.

Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos

DeMicco, A., Cooper, K. R., Richardson, J. R., White, L. A. — Tue, 27 Oct 2009 12:28:43 PDT

Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based of the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, 3 type I compounds (permethrin, resmethrin, and bifenthrin) and 3 type II compounds (deltamethrin, cypermethrin, and -cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC₅₀, permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with Diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

Effects of Pioglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Agonist, on the Urine and Urothelium of the Rat

Sun, 25 Oct 2009 19:27:49 PDT

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which belong to the nuclear receptor superfamily. Some PPAR agonists, such as pioglitazone, and dual PPAR/PPAR agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyurindine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPAR agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms

Makaji, E., Trambitas, C. S., Shen, P., Holloway, A. C., Crankshaw, D. J. — Sun, 25 Oct 2009 19:27:48 PDT

We have evaluated the use of a panel of six fluorogenic cytochrome P450 substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin, 7-benzyloxy-4-(trifluoromethyl)-coumarin ,7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifuoromethyl)-coumarin and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male rat liver were characterized, their sensitivities to fifteen putative inhibitors were determined and compared to similar experiments using nine different cDNA-expressed rat CYPs. Inhibitory profiles of the substrates in microsomes were different from each other, with some overlap, suggesting that each substrate is to some extent biotransformed by a different CYP isoform. Ketoconazole and clotrimazole were non-selective inhibitors while ticlopidine selectively inhibited biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin. CYP 2A1 did not biotransform any of the substrates and CYP 2E1 was insensitive to all the inhibitors tested. Some inhibitors did not affect the biotransformation of the fluorogenic substrates by cDNA-expressed isoforms as predicted by their effects on conventional substrates, for example chlorzoxazone and diethyldithiocarbamate were inactive against CYP2E1, and CYP2C6 was not inhibited by sulfaphenazole. When results in microsomes and cDNA-expressed CYPs were compared, only the majority of the biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin by microsomes could be assigned with full confidence to a specific CYP isoform, namely CYP2D2. Nevertheless different inhibitory profiles of the substrates indicate that the panel will be useful for rapid functional quantification of global CYP activity in rats under different experimental conditions. Our results also demonstrate the inappropriateness of extrapolating inhibitory data between conventional and fluorogenic CYP substrates.

Characterization of Peroxisome Proliferator-Activated Receptor {alpha} (PPAR{alpha}) - Independent Effects of PPAR{alpha} Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor

Thu, 22 Oct 2009 05:14:06 PDT

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR). Recent studies indicate that the plasticizer di-2-ethylhexyl phthalate (DEHP) increased the incidence of liver tumors in PPAR-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPAR but dependent on other nuclear receptors, including the constitutive activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate and DEHP in rat liver and DEHP in mouse liver induced genes including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and RT-PCR in the livers of DEHP-treated wild-type, PPAR-null and CAR-null mice demonstrated that 1) most (~94%) of the transcriptional changes induced by DEHP were PPAR-dependent, 2) many PPAR-independent genes overlapped with those regulated by PB, 3) induction of genes Cyp2b10, Cyp3a11 and metallothionine-1 by DEHP was CAR-dependent but PPAR-independent and 4) induction of a number of genes (Cyp8b1, Gstm4, Gstm7) was independent of both CAR and PPAR. Our results indicate that exposure to PPAR activators including DEHP activate multiple nuclear receptors in the rodent liver.

Mechanisms Mediating the Vesicant Actions of Sulfur Mustard after Cutaneous Exposure

Thu, 15 Oct 2009 06:22:51 PDT

Sulfur mustard, a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to sulfur mustard involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. Sulfur mustard is a bifunctional alkylating agent which reacts with many targets including lipids, proteins and DNA, forming both intra- and intermolecular cross-links. Despite the relatively non-selective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of sulfur mustard-induced cytotoxicity, and the intracellular and extracellular targets of this vesicant. In this review, the effects of sulfur mustard exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for sulfur mustard poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

Oxidative Stress Caused by Ozone Exposure Induces Loss of Brain Repair in The Hippocampus of Adult Rats

Thu, 15 Oct 2009 06:22:49 PDT

Oxidative stress is involved in many neurodegenerative diseases. Chronic ozone exposure causes a secondary increase of reactive oxygen species, which cause an oxidative-stress state in the organism. Ozone is one of the main components of photochemical pollution. Our purpose was to test that oxidative stress caused by chronic low doses of ozone, by itself, alters adult neurogenesis and causes progressive neurodegeneration in the hippocampus, which actions lead to the loss of brain plasticity in the mature central nervous system of rats. Animals were exposed to an ozone-free air stream and for 15, 30, 60, and 90 days to low doses of ozone to cause oxidative stress. Each group was then tested by 1) a spectrophotometer test to quantify lipid peroxidation levels, 2) immunohistochemistry testing against doublecortine, Neu-N, p53, microglia, and glial fibrillary-acidic protein (GFAP), 3) western blot tests for doublecortine and Neu-N, and 4) a one-trial passive avoidance test. Our results indicated that ozone causes an increase of lipid peroxidation levels, morphological changes in the nucleus and the cytoplasm, and cell swelling in neurons. The western blot shows a decrease for Neu-N and doublecortine. Activated and later phagocytic microglia and an increased number of astrocytes were found. There was a memory deficiency positively related to the amount of ozone exposure. These alterations suggest that oxidative stress caused by low doses of ozone causes dysregulation of inflammatory processes, progressive neurodegeneration, chronic loss of brain repair in the hippocampus, and brain plasticity changes in the rat analogous to those seen in Alzheimer's disease.

Arsenic (+3 Oxidation State) Methyltransferase and the Methylation of Arsenicals in the Invertebrate Chordate Ciona intestinalis

Thu, 15 Oct 2009 06:22:47 PDT

Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+3 oxidation state) methyltransferase (As3mt), yielding mono- , di- , and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation, a comparative genomic approach focusing on the invertebrate chordate Ciona intestinalis, was used. Bioinformatic analyses identified an As3mt gene in the C. intestinalis genome. Constitutive As3mt RNA expression was observed in heart, branchial sac and gastrointestinal tract. Adult animals were exposed to zero or 1 ppm of iAs for 1 or 5 days. Steady-state As3mt RNA expression in the gastrointestinal tract was not modulated significantly by 5 days of exposure to iAs. Tissue levels of iAs and its methylated metabolites were determined by hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometry. At either time point, exposure to iAs significantly increased concentrations of iAs and its methylated metabolites in tissues. After 5 days of exposure, total speciated arsenic concentrations were highest in branchial sac (3705 ng/g) followed by heart (1019 ng/g) and gastrointestinal tract (835 ng/g). At this time point, the sum of the speciated arsenical concentrations in gastrointestinal tract and heart equaled or exceeded that of iAs; in branchial sac, iAs was the predominant species present. Ciona intestinalis metabolizes iAs to its methylated metabolites, which are retained in tissues. This metabolic pattern is consistent with the presence of an As3mt ortholog in its genome and constitutive expression of the gene in prominent organs, making this basal chordate a useful model to examine the evolution of arsenic detoxification.

Decreased expression of the voltage-dependent anion channel in differentiated PC-12 and SH-SY5Y cells following Low-level Pb exposure

Prins, J. M., Park, S., Lurie, D. I. — Mon, 12 Oct 2009 04:29:50 PDT

Lead (Pb) has been shown to disrupt cellular energy metabolism which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5µM and 10µM Pb on VDAC expression in-vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response, but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time RT-PCR demonstrated a significant decrease in mRNA levels for the VDAC 1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5µM and 10µM Pb reducesVDAC transcription and expression and is associated with reduced cellular ATP levels.

Proteomic analysis of hepatic tissue of zebrafish (Danio rerio) experimentally exposed to chronic microcystin-LR

Wang, M., Chan, L. L., Si, M., Hong, H., Wang, D. — Mon, 12 Oct 2009 04:29:47 PDT

Microcystin-LR (MCLR) is the most toxic and most frequently encountered hepatotoxin in the aquatic environment. This study investigated the protein profiles of zebrafish (Danio rerio) livers chronically exposed to MCLR concentrations (2 or 20 µg/l) using the proteomic approach as well as cell ultrastructure, protein phosphatase (PP) activity, protein phosphatase 2A (PP2A) abundance and toxin content analysis of the hepatic tissue. The results showed that, after 30 d exposure, the presence of MCLR strikingly enhanced toxin accumulation and the PP activity in zebrafish livers. However, the PP2A amounts were independent of toxin treatments. MCLR caused a noticeable damage to liver ultrastructure, a widespread swelling in the rough endoplasmatic reticulum and mitochondria was observed in the MCLR-exposed hepatocytes, and a honeycomb-like structure was formed in the treated nucleoli. Comparison of two-dimensional electrophoresis (2-DE) protein profiles of MCLR-exposed and non-exposed zebrafish livers revealed that the abundance of 22 proteins, measured by 2-DE, was remarkably altered in response to toxin exposure. These proteins were involved in cytoskeleton assembly, macromolecule metabolism, oxidative stress and signal transduction, indicating that MCLR toxicity in fish liver is complex and diverse. Thus, proteomics provides a new insight into MCLR toxicity; that chronic toxicity of MCLR is different from acute toxicity; and we speculate that the reactive oxygen species pathway might be the main toxic pathway instead of the PP one. Moreover, even a low concentration of MCLR in water could significantly interrupt cellular processes, and more care should be taken in determining the criterion for MCLR content in drinking water.

Subchronic 13-week Inhalation Exposure of Rats to Multiwalled Carbon Nanotubes: Toxic Effects are determined by Density of Agglomerate Structures, not fibrillar Structures

Pauluhn, J. — Mon, 12 Oct 2009 04:29:43 PDT

Wistar rats were nose-only exposed to Multi-walled Carbon Nanotubes (MWCNT, Baytubes^®) in a subchronic 13-week inhalation study. The focus of study was on respiratory tract and systemic toxicity, including analysis of MWCNT biokinetics in the lungs and lung-associated lymph nodes (LALNs). The time-course and concentration-dependence of pulmonary effects were examined by bronchoalveolar lavage (BAL), and histopathology up to 6 months postexposure. Particular emphasis was directed to the comparative characterization of MWCNT structures prior to and after micronization and dry powder dispersion into inhalation chambers. These determinations were complemented by additional analyses in digested BAL-cells. Animals were exposed on 6 hrs/day, five days per week for 13 consecutive weeks to 0, 0.1, 0.4, 1.5, and 6 mg/m³. The subchronic exposure to respirable solid aerosols of MWCNT was tolerated without effects suggestive of systemic toxicity. Kinetic analyses demonstrated a markedly delayed clearance of MWCNT from lungs at overload conditions. Translocation into LALNs occurred at 1.5 and 6 mg/m³ and required at least 13 weeks of study to become detectable. At these exposure levels the lung and LALN weights were significantly increased. Sustained elevations in BAL PMNs and soluble collagen occurred at these concentrations with borderline effects at 0.4 mg/m³. Histopathology revealed principal exposure-related lesions at 0.4 mg/m³ and above in the upper respiratory tract (goblet cell hyper- and/or metaplasia, eosinophilic globules, focal turbinate remodeling) and the lower respiratory tract (inflammatory changes in the bronchiolo-alveolar region, increased interstitial collagen staining). Granulomatous changes and a time-dependent increase of a bronchiolio-alveolar hyperplasia occurred at 6 mg/m³. All endpoints examined were unremarkable at 0.1 mg/m³ (NOAEL). In summary, this study demonstrates that the induced pathological changes are consistent with overload-related phenomena. Hence, the etiopathological sequence of inflammatory events caused by this type of MWCNT appears to be related to the high displacement volume of the low-density MWCNT-assemblage structure rather than to any yet ill-defined intrinsic toxic property. Thus, the hypothesis of study is verified, namely common denominators between carbon black and MWCNT do exist.

Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-jun-N-terminal kinase activation in cultured neurons

Vale, C., Nicolaou, K. C., Frederick, M. O., Vieytes, M. R., Botana, L. M. — Thu, 08 Oct 2009 10:58:29 PDT

Azaspiracids are a group of marine toxins recently described that currently includes 20 analogues. Not much is known about their mechanism of action, although the predominant analogue in nature, azaspiracid-1 (AZA-1) targets several organs in vivo, including the central nervous system and exhibits high neurotoxicity in vitro. Azaspiracid distribution is increasing globally with mussels being most widely implicated in azaspiracid-related food poisoning events and human poisoning by azaspiracids has emerged as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for azaspiracid-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro (div), however it did not modify membrane potential in mature neurons. Furthermore, in immature cells AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50 mM K⁺. Preincubation of the neurons with 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-Acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride or ouabain before addition of AZA-1 decreased the AZA-1 induced neurotoxicity and the increase in phosphorylated JNK caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Expression of Tyrosine Hydroxylase Increases the Resistance of Human Neuroblastoma Cells to Oxidative Insults

Thu, 08 Oct 2009 10:58:29 PDT

In this study we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH+TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine, when compared to wild type SH-SY5Y cells (SH cells). SH+TH cells exhibit increased levels of dopamine compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10 – 100 µM) for 24 hours caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH+TH cells when compared to the SH cells. The SH+TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activity, while catalase activity was unchanged. Our data suggests that a preconditioning-like mechanism linked to higher dopamine levels increased the resistance of SH+TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.

Reduction of glutamatergic neurotransmission by prolonged exposure to dieldrin involves NMDA receptor internalization and metabotropic glutamate receptor 5 down-regulation

Briz, V., Galofre, M., Sunol, C. — Wed, 07 Oct 2009 11:02:06 PDT

Dieldrin was previously used as a pesticide. Although its use has been discontinued, humans are still exposed to it due to its high environmental persistence and because it accumulates in the adipose tissue of animals. Acute exposure to dieldrin provokes convulsions, due to its antagonism on the GABA_A receptor. However little is known about the effects of low chronic exposure to this pollutant. In the present work we use primary cultures of cortical neurons to study the mechanisms involved in the toxic action of dieldrin. We found that 2 and 6 days in vitro (DIV) exposure to a subcytotoxic concentration (60nM) of dieldrin reduced the increase in intracellular calcium concentration ([Ca²⁺]_i) and the excitotoxicity caused by glutamate. Exposure to dieldrin for 6 DIV induced NMDA receptor (NMDAR) internalization and reduced metabotropic glutamate receptor 5 (mGLUR5) levels. Double immunostaining for NMDAR and mGLUR5 showed that these receptors lose colocalization on the cell membrane in neurons treated with dieldrin. No changes were observed in receptor functionalities or receptor levels after 2 DIV of exposure to dieldrin. However, the increase in [Ca²⁺]_i induced by coactivation of NMDAR and mGLUR5 was significantly reduced. Thus, a functional interaction between the two receptors seems to play an important role in glutamate-induced excitotoxicity. We confirm that permanent blockade of the GABA_A receptor by this persistent pesticide triggers adaptive neuronal changes consisting of a reduction of glutamatergic neurotransmission. This might explain the cognitive and learning deficits observed in animals after chronic treatment with dieldrin.

In Utero Exposure to Benzene Disrupts Fetal Hematopoietic Progenitor Cell Growth Via Reactive Oxygen Species

Badham, H. J., Winn, L. M. — Wed, 07 Oct 2009 11:02:06 PDT

It is hypothesized that the increasing incidence of childhood leukemia may be due to in utero exposure to environmental pollutants such as benzene, but the mechanisms involved remain unknown. We hypothesize that reactive oxygen species (ROS) contribute to the deregulation of fetal hematopoiesis caused by in utero benzene exposure. To evaluate this hypothesis, pregnant C57Bl/6N mice were exposed to benzene or PEG-catalase (antioxidative enzyme) and benzene. Colony formation assays on fetal liver cells were performed to measure erythroid and myeloid progenitor cell growth potential. The presence of ROS in CD117⁺ fetal liver cells was measured by flow cytometric analysis. Oxidative cellular damage was assessed by Western blot analysis of 4-hydroxynonenol and nitrotyrosine products, as well as reduced to oxidized glutathione ratios. Alterations in the redox sensitive signaling pathway NF-B were measured by Western blot analysis of IB- protein levels in fetal liver tissue. Results: In utero exposure to benzene caused a significant increase in ROS production and significantly altered fetal liver erythroid and myeloid colony numbers, but did not increase the levels of 4-HNE or nitrotyrosine products or alter reduced to oxidized glutathione ratios. However, in utero exposure to benzene did cause a significant decrease in fetal liver IB- protein levels suggesting activation of the NF-B pathway. Benzene-induced ROS formation, abnormal colony growth, and decreased IB- levels were all abrogated by pre-treatment with PEG-catalase. These results suggest that ROS play a key role in the development of in utero-initiated benzene toxicity potentially through disruption of hematopoietic cell signaling pathways.

Cytotoxicity and Regenerative Proliferation as the Mode of Action for Diuron-Induced Urothelial Carcinogenesis in the Rat

Wed, 07 Oct 2009 11:02:05 PDT

Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2,500 ppm, about 130 mg/kg of body weight) either with or without NH₄Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25 or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron+NH₄Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron and in diuron+NH₄Cl treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with CHO cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

The Role of Hypoxia in 2-Butoxyethanol-induced Hemangiosarcoma

Wed, 07 Oct 2009 11:02:04 PDT

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network^TM Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow, liver, and spleen of mice exposed to a single dose (4h) or 7 daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased Epo signaling and erythropoiesis in the spleen and bone marrow, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe^TM) demonstrated that tissue hypoxia was present in the spleen and bone marrow. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE-induced hemangiosarcoma in mice.

P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite and nitrate developmental toxicity

Solari, M., Paquin, J., Ducharme, P., Boily, M. — Tue, 06 Oct 2009 07:17:10 PDT

Atrazine and nitrogenous fertilizers are agrochemical contaminants frequently detected in water systems in North America. Several studies reported their ability to affect amphibian and mammalian development. Retinoids, supplied in the diet or synthesized by cells, are essential to embryogenesis. Disturbance of their homeostasis may lead to teratogenic effects. Retinoic acid (RA) is a major retinoid regulator of cell proliferation and differentiation. Previous studies reported alterations of retinoid stores in bullfrogs of Yamaska River subwatersheds (Québec, Canada), a region of intensive agricultural activities associated with atrazine, nitrate and nitrite contaminants. These contaminants could affect RA metabolism and RA-mediated processes. Mouse P19 embryonic stem cells, which can differentiate to neurons in response to RA, were used to test this hypothesis. Cells were cultured in the absence or presence of contaminants during neuroinduction with RA, and assayed by flow cytometry for expression of SSEA1 (embryonic marker) and βIII-tubulin (neuronal marker). Cell cultures were also analysed for RA metabolism by HPLC. Downregulation of SSEA1 paralleled βIII-tubulin upregulation in a RA concentration-dependent manner. Atrazine, nitrate and nitrite did not affect differentiation at environmentally-encountered micromolar concentrations. However, low molar nitrite prevented RA-induced SSEA1 downregulation, and decreased βIII-tubulin appearance. Decreased cell viability/proliferation accompanied these differentiation effects. P19 cells metabolized RA to polar retinoids. RA metabolism was not affected at any concentration of atrazine, nitrate or nitrite. Environmentally-relevant levels of these contaminants thus had no gross effect on neurodifferentiation and RA catabolism of embryonic stem cells. P19 cell-based bioassays may provide valuable tools in monitoring developmental toxicity.

Use of gene expression of neural markers in cultured neural cells to identify developmental neurotoxicants

Sunol, C. — Tue, 06 Oct 2009 07:17:09 PDT

Rapamycin inhibits yeast nucleotide excision repair independently of Tor kinases

Limson, M. V., Sweder, K. S. — Mon, 05 Oct 2009 09:01:43 PDT

The yeast target of rapamycin kinases, Tor1 and Tor2, belong to the phosphatidylinositol 3-kinase related family of proteins, which are involved in the cellular response to DNA damage and changes in nutrient conditions. In contrast to yeast, many eukaryotes possess a single Tor kinase. Regardless of the number of Tor kinases in an organism, two distinct complexes involving Tor proteins exist in eukaryotes, TORC1 and TORC2. The yeast TORC1, containing Tor1 or Tor2, is sensitive to the antibiotic rapamycin. The yeast TORC2 is insensitive to rapamycin. We examined the influence of rapamycin treatment upon yeast transcription-coupled nucleotide excision repair in a gene transcribed by RNA polymerase II. We also examined tor mutants for their ability to perform transcription-coupled repair in the absence or presence of rapamycin. Ostensibly lacking TORC1 and TORC2 function, a tor1tor2^ts mutant grown at the nonpermissive temperature exhibited similar rates of repair as the wild-type strain. However, repair of both strands in genes decreases in the wild-type strain and the tor1tor2^ts mutant exposed to rapamycin. Rapamycin may be inhibiting DNA repair independently of the Tor kinases. In yeast, FPR1 encodes the rapamycin-binding protein Fpr1 that inhibits the TORC1 kinase in the presence of rapamycin. Fap1 competes with rapamycin for Fpr1 binding. Deletion of the FPR1 or FAP1 gene abolishes the inhibitory effect of rapamycin on repair. Thus, the decreased repair observed following rapamycin treatment is independent of TORC1/2 function and likely due to a function of Fap1. We suggest that Fap1 and peptidyl prolyl isomerases, particularly Fpr1, function in the cellular response to genotoxic stress. Our findings have clinical implications for genetic toxicities associated with genotoxic agents when co-administered with rapamycin.

Differential impact of diesel particle composition on pro-allergic dendritic cell function

Sun, 04 Oct 2009 22:28:24 PDT

Diesel exhaust particles (DEP) were described as potent adjuvant in the induction and maintenance of allergic diseases suggesting that they might play a role in the increase of allergic diseases in the industrialized countries. However, the cellular basis by which these particles enhance allergic immune responses is still a matter of debate.

Thus, we exposed immature murine bone marrow-derived dendritic cells (BMDC) to different particles or particle-associated organic compounds in the absence or presence of the maturation stimuli lipopolysachharide and analyzed the cellular maturation, viability, and cytokine production. Furthermore, we monitored the functionality of particle-exposed BMDC to suppress B cell isotype switching to IgE.

Only highly polluted DEP (standard reference material SRM1650a) but not particle-associated organic compounds or less polluted DEP from modern diesel engines were able to modulate the dendritic cell phenotype. SRM1650a particles significantly suppressed LPS-induced IL-12p70 production in murine BMDC, whereas cell surface marker expression was not altered. Furthermore, SRM1650a-exposed immature BMDC lost the ability to suppress IgE isotype switch in B cells.

This study revealed that highly polluted DEP not only interfere with dendritic cell maturation but additionally with dendritic cell function, thus suggesting a role in Th2 immune deviation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Fibroblast Growth Factor 10-Induced Prostatic Bud Formation in Mouse Urogenital Sinus

Vezina, C. M., Hardin, H. A., Moore, R. W., Allgeier, S. H., Peterson, R. E. — Sun, 04 Oct 2009 22:28:23 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dorsalizes the pattern of prostatic buds developing from the urogenital sinus (UGS) of male fetal mice, causing some buds to form in inappropriate positions while blocking formation of others. This teratogenic TCDD action significantly reduces prostate main duct number and causes ventral prostate agenesis in exposed males. The purpose of this study was to determine whether inhibition of fibroblast growth factor 10 (FGF10) signaling is mechanistically linked to mouse prostatic budding impairment by TCDD. In utero TCDD exposure induced aryl hydrocarbon receptor (AHR)-responsive cytochrome P450 1b1 mRNA in ventral UGS regions where Fgf10 and FGF receptor 2 (Fgfr2) mRNA were expressed and where budding was most severely inhibited by TCDD. However, TCDD exposure did not reduce Fgf10 or Fgfr2 mRNA abundance in the UGS or alter their distribution. Addition of FGF10 protein to UGS organ culture media increased the abundance of UGS basal epithelial cells immuno-positive for phosphorylated extracellular signal-regulated kinase (ERK). FGF10 also increased the number of bromodeoxyuridine (BrdU)-labeled UGS epithelial cells and increased the number of prostatic buds formed per UGS. Addition of TCDD to UGS organ culture media did not alter FGF10-induced ERK activation in UGS basal epithelium but prevented FGF10-induced BrdU incorporation and blocked FGF10-induced prostatic bud formation. These results identify basal UGE cells as the key site of FGF10 action during fetal prostate development and suggest that TCDD likely acts downstream of FGFR2 and ERK to restrict UGS epithelial cell proliferation and prevent prostatic bud formation.

Suppression of Insulin-Like Growth Factor Acid-Labile Subunit (IGFALS) Expression - A Novel Mechanism for Deoxynivalenol-Induced Growth Retardation

Amuzie, C. J., Pestka, J. J. — Sun, 04 Oct 2009 22:28:22 PDT

Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species. While growth retardation is used as a basis for regulating DON levels in human food, the underlying mechanisms remain poorly understood. Oral exposure of mice to DON rapidly induces multi-organ expression of proinflammatory cytokines and this is followed by upregulation of several suppressors of cytokine signaling (SOCS), some of which are capable of impairing growth hormone (GH) signaling. The purpose of this study was to test the hypothesis that impairment of the GH axis precedes DON-induced growth retardation in the mouse. Subchronic dietary exposure of young (4-wk old) mice to DON (20 ppm) over a period of 2 to 8 wk was found to: (1) impair weight gain, (2) result in a steady-state plasma DON concentration (40-60 ng/ml), (3) downregulate hepatic insulin-like growth factor acid labile subunit (IGFALS) mRNA expression and (4) reduce circulating insulin-like growth factor 1 (IGF1) and IGFALS levels. Acute oral exposure to DON at 0.5-12.5 mg/kg bw markedly suppressed hepatic IGFALS mRNA levels within 2 h in a dose-dependent fashion whereas 0.1 mg/kg bw was without effect. DON-induced IGFALS mRNA upregulation occurred both with and without exogenous GH treatment. These latter effects co-occurred with robust hepatic SOCS3 upregulation. Taken together, these data suggest that oral DON exposure perturbs GH axis by suppressing two clinically relevant growth-related proteins, IGFALS and IGF1. Both have potential to serve as biomarkers of effect in populations exposed to this common foodborne mycotoxin.

Diesel exhaust particles in the lung aggravate experimental acute renal failure

Nemmar, A., Al-Salam, S., Zia, S., Yasin, J., Al Husseni, I., Ali, B. H. — Thu, 01 Oct 2009 04:06:47 PDT

Inhaled particles are associated with pulmonary and extrapulmonary effects. Also, acute renal failure (ARF) is associated with increased mortality, related to pulmonary complications. Here, we tested the possible potentiating effect of diesel exhaust particles (DEP) in an animal model of ARF induced by a single intraperitoneal injection of cisplatin (CP, 6 mg/kg) in rats. Six days later, the rats were intratracheally (i.t.) instilled with either DEP (0.5 or 1 mg/kg) or saline (control), and renal, systemic and pulmonary variables were studied 24 h thereafter. CP increased the serum concentrations of urea, creatinine, and reduced glutathione (GSH) concentration and superoxide dismutase activity in renal cortex. CP caused renal tubular necrosis, and increased urine volume, protein concentrations and N-acetyl-β-D-glucosaminidase (NAG) activity, and decreased urine osmolality. The combination of DEP and CP aggravated the CP-induced effects on serum urea and creatinine, urine NAG activity, and renal GSH. The arterial O₂ saturation and PO₂ were significantly decreased in CP + DEP versus CP + saline, and CP+DEP versus DEP. The number of platelets was reduced in DEP compared to saline-treated rats, and CP + DEP versus DEP alone or CP + saline. Increases in macrophage and neutrophils numbers in bronchoalveolar lavage were found in DEP versus saline group, and CP + DEP versus CP. Histopathological changes in lungs of DEP –treated rats were aggravated by the combination of CP + DEP. These included marked interstitial cell infiltration and congestion. We conclude that the presence of DEP in the lung aggravated the renal, pulmonary and systemic effects of CP-induced ARF.

Capture Compound Mass Spectrometry (CCMS) Sheds Light on the Molecular Mechanisms of Liver Toxicity of two Parkinson Drugs

Sat, 26 Sep 2009 04:01:04 PDT

Capture Compound Mass Spectrometry (CCMS) is a novel technology that helps to understand the molecular mechanism of the mode of action of small molecules. The Capture Compounds are trifunctional probes: a selectivity function (the drug) interacts with the proteins in a biological sample, a reactivity function (phenylazide) irreversibly forms a covalent bond, and a sorting function (biotin) allows the captured protein(s) to be isolated for mass spectrometric analysis.

Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinson's disease. We aimed to understand the molecular basis for the difference of both drugs in respect to side effects. Using Capture Compounds with these drugs as selectivity functions we were able to unambiguously and reproducibly isolate and identify their known target COMT. Tolcapone Capture Compounds captured five times more proteins than entacapone Capture Compounds. Moreover, tolcapone Capture Compounds isolated mitochondrial and peroxisomal proteins. The major tolcapone – protein interactions occurred with components of the respiratory chain and of the fatty acid β-oxidation. Previously reported symptoms in tolcapone-treated rats suggested that tolcapone might act as decoupling reagent of the respiratory chain (Haasio et al).

Our results demonstrate that CCMS is an effective tool for the identification of a drug's potential off-targets. It fills a gap in currently used in-vitro screens for drug profiling that do not contain all of the toxicologically relevant proteins. Thereby CCMS has the potential to fill a technological need in drug safety assessment and helps to reengineer or to reject drugs at an early pre-clinical stage.

Approaches for Assessing Risks to Sensitive Populations: Lessons Learned from Evaluating Risks in the Pediatric Population

Mon, 21 Sep 2009 13:15:39 PDT

Assessing the risk profiles of potentially sensitive populations requires a ‘tool chest’ of methodological approaches to adequately characterize and evaluate these populations. At present, there is an extensive body of literature on methodologies that apply to the evaluation of the pediatric population. The HESI Subcommittee on Risk Assessment of Sensitive Populations evaluated key references in the area of pediatric risk to identify a spectrum of methodological approaches. These approaches are considered in this manuscript for their potential to be extrapolated for the identification and assessment of other sensitive populations. Recommendations as to future research needs and/or alternate methodological considerations also are made.

The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers -functional relevance and a hypothesis on innate reactions to skin sensitizers

Natsch, A. — Fri, 18 Sep 2009 23:12:02 PDT

With the tight deadlines set both by the public and by regulatory authorities to replace animal tests for toxicological endpoints relevant to the development of cosmetic products, a large number of research projects have recently focused on cellular endpoints affected by skin sensitizing compounds. The general aim stated in these projects was to find ‘markers’ for skin sensitizers, be it at the level of the transcriptome or at the protein level. Rather then talking of ‘cellular markers’, the new paradigm ‘Toxicity testing in the 21th century’ formulated by the National Academy of Sciences in the United States focuses on ‘Toxicity pathways’. A specific ‘marker’ for any given toxicological endpoint can only exist, if specific toxicity pathways, comprising specific sensors, are linked to this endpoint. In the context of skin sensitization, one has to ask whether there is an innate cellular signaling pathway activated by skin sensitizers. Here a significant body of evidence, mainly accumulated in the last 20 months, is reviewed, indicating that indeed the Nrf2-Keap1-ARE regulatory pathway is such a toxicity pathway activated by cysteine-reactive skin sensitizers. Whereas first indications on the in vivo relevance are available, key questions remain open and can now specifically be addressed. A minority of sensitizers, more specifically reacting with lysine residues, appears not to activate the Nrf2-Keap1-ARE pathway, and might trigger yet another unknown toxicity pathway.

Ligand Activation of Peroxisome Proliferator-Activated Receptor-{beta}/{delta} (PPAR{beta}/{delta}) and Inhibition of Cyclooxygenase 2 (COX2) Enhances Inhibition of Skin Tumorigenesis

Bility, M. T., Zhu, B., Kang, B. H., Gonzalez, F. J., Peters, J. M. — Fri, 11 Sep 2009 05:26:14 PDT

Ligand activation of peroxisome proliferator-activated receptor-β/ (PPARβ/) and inhibition of cyclooxygenase-2 (COX2) activity by non-steroidal anti-inflammatory drugs (NSAID) can both attenuate skin tumorigenesis. There is also evidence that attenuation of skin tumorigenesis by inhibition of COX2 activity occurs through PPARβ/–independent mechanisms. The present study examined the hypothesis that combining ligand activation of PPARβ/ with inhibition of COX2 activity will cooperatively inhibit chemically-induced skin tumor progression using both in vivo and ex vivo models. A two-stage chemical carcinogenesis bioassay was performed in wild-type and Pparβ/-null mice. After twenty-two weeks, cohorts of both mouse lines were divided into four experimental groups: 1) control, 2) topical application of the PPARβ/ ligand GW0742, 3) dietary administration of the COX2 inhibitor nimesulide, or 4) both GW0742 and nimesulide. Ligand activation of PPARβ/ did not influence skin tumor progression while a modest decrease in skin tumor multiplicity was observed with dietary nimesulide. Interestingly, the combined treatment of GW0742 and nimesulide increased the efficacy of the decrease in papilloma multiplicity for six weeks in wild-type mice, but this effect was not found at later timepoints and was not found in similarly treated Pparβ/-null mice. Neoplastic keratinocyte lines cultured with GW0742 and nimesulide also exhibited enhanced inhibition of cell proliferation coincident with increased expression of Keratin mRNAs. Results from these studies support the hypothesis that combining ligand activation of PPARβ/ with inhibition of COX2 activity can inhibit chemically-induced skin tumor progression by modulating differentiation.

mRNA expression is a relevant tool to identify developmental neurotoxicants using an in vitro approach

Mon, 03 Aug 2009 03:17:07 PDT

So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Since the current developmental neurotoxicity (DNT) guidelines (OECD TG 426) are based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritise chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid and tri-methyl tin chloride) for different time periods. A significant down-regulation of the mRNA level for the neuronal markers (NF-68, NF-200, NMDA-R and GABAA-R) was observed after exposure to methyl mercury chloride, valproic acid and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (GFAP and S100β) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP while the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation.

OGG1 and MYH are involved in the incision of trivalent arsenical-induced DNA adducts

Pu, Y.-S., Jan, K.-Y., Wang, T.-C., Wang, A. S. S., Gurr, J.-R. — Wed, 29 Nov 2006 06:47:56 PST

Since trivalent arsenicals are known to induce oxidative DNA damage in human cells, we asked if they induce other types of DNA damage and how these DNA damages are repaired. Treatment of human promyelocytic leukemia NB4 cells with 0.5 µM As₂O₃ for 30 min induced no DNA breaks, as analyzed by a standard comet assay. However, breaks were detected if these cells were then digested with endonuclease III (EnIII), formamidopyrimidine-DNA glycosylase (Fpg), or a nuclear extract (NE) of NB4 cells. Using either H₂O₂-Fe treated nuclei or As₂O₃-treated cells, digestion with either NE or EnIII+Fpg generated the same amount of breaks, and subsequent treatment with EnIII+Fpg resulted in no increase in breaks in NE-digested cells and vice versa. The human cell lines, defective in nucleotide excision protein, such as XPA, XPD, and XPG, excised UVC-induced adducts less rapidly than normal fibroblasts, but excised As₂O₃-adducts at the same rate as the normal cells. Immunodepletion of the NE with antibody against OGG1 or MYH decreased the incision of DNA adducts induced by As₂O₃, NaAsO₂, monomethylarsonic acid, and dimethylarsinic acid, while antibodies against XPA, XPB, XPD, XPF, or XPG, did not. These results suggest that these trivalent arsenicals induce the formation of only oxidative DNA adducts and that OGG1 and MYH are involved in these incision processes.