Toxicological Sciences - recent issues

2009-06-15

COVER

2009-06-15

Editorial Board

2009-06-15

SUBSCRIPTIONS

2009-06-15

Nitric Oxide Synthase: "Enzyme Zero" in Air Pollution-Induced Vascular Toxicity

Campen, M. J. — 2009-06-15

Chiral Toxicology: It's the Same Thing...Only Different

Smith, S. W. — 2009-06-15

Chiral substances possess a unique architecture such that, despite sharing identical molecular formulas, atom-to-atom linkages, and bonding distances, they cannot be superimposed. Thus, in the environment of living systems, where specific structure-activity relationships may be required for effect (e.g., enzymes, receptors, transporters, and DNA), the physiochemical and biochemical properties of racemic mixtures and individual stereoisomers can differ significantly. In drug development, enantiomeric selection to maximize clinical effects or mitigate drug toxicity has yielded both success and failure. Further complicating genetic polymorphisms in drug disposition, stereoselective metabolism of chiral compounds can additionally influence pharmacokinetics, pharmacodynamics, and toxicity. Optically pure pharmaceuticals may undergo racemization in vivo, negating single enantiomer benefits or inducing unexpected effects. Appropriate chiral antidotes must be selected for therapeutic benefit and to minimize adverse events. Enantiomers may possess different carcinogenicity and teratogenicity. Environmental toxicology provides several examples in which compound bioaccumulation, persistence, and toxicity show chiral dependence. In forensic toxicology, chiral analysis has been applied to illicit drug preparations and biological specimens, with the potential to assist in determination of cause of death and aid in the correct interpretation of substance abuse and "doping" screens. Adrenergic agonists and antagonist, nonsteroidal anti-inflammatory agents, SSRIs, opioids, warfarin, valproate, thalidomide, retinoic acid, N-acetylcysteine, carnitine, penicillamine, leucovorin, glucarpidase, pesticides, polychlorinated biphenyls, phenylethylamines, and additional compounds will be discussed to illustrate important concepts in "chiral toxicology."

Safety Assessment of Biotechnology Products for Potential Risk of Food Allergy: Implications of New Research

Selgrade, M. K., Bowman, C. C., Ladics, G. S., Privalle, L., Laessig, S. A. — 2009-06-15

Food allergy is a potential risk associated with use of transgenic proteins in crops. Currently, safety assessment involves consideration of the source of the introduced protein, in silico amino acid sequence homology comparisons to known allergens, physicochemical properties, protein abundance in the crop, and, when appropriate, specific immunoglobulin E binding studies. Recently conducted research presented at an International Life Sciences Institute/Health and Environmental Sciences Institute–hosted workshop adds to the scientific foundation for safety assessment of transgenic proteins in five areas: structure/activity, serum screening, animal models, quantitative proteomics, and basic mechanisms. A web-based tool is now available that integrates a database of allergenic proteins with a variety of computational tools which could be used to improve our ability to predict allergenicity based on structural analysis. A comprehensive strategy and model protocols have been developed for conducting meaningful serum screening, an extremely challenging process. Several animal models using oral sensitization with adjuvant and one dermal sensitization model have been developed and appear to distinguish allergenic from non-allergenic food extracts. Data presented using a mouse model suggest that pepsin resistance is indicative of allergenicity. Certain questions remain to be addressed before considering animal model validation. Gel-free mass spectrometry is a viable alternative to more labor-intensive approaches to quantitative proteomics. Proteomic data presented on four nontransgenic varieties of soy suggested that if known allergen expression in genetically modified crops falls within the range of natural variability among commercial varieties, there appears to be no need to test further. Finally, basic research continues to elucidate the etiology of food allergy.

Toxicity Testing in the 21st Century: A View from the Pharmaceutical Industry

MacDonald, J. S., Robertson, R. T. — 2009-06-15

Compensatory Induction of Liver Efflux Transporters in Response to ANIT-Induced Liver Injury Is Impaired in FXR-Null Mice

Cui, Y. J., Aleksunes, L. M., Tanaka, Y., Goedken, M. J., Klaassen, C. D. — 2009-06-15

Alpha-naphthyl isothiocyanate (ANIT) is a hepatotoxicant that produces acute intrahepatic cholestasis in rodents. Farnesoid X receptor (FXR) and pregnane X receptor (PXR) are two major bile acid sensors in liver. The purpose of this study was to characterize the regulation of hepatic transporters by FXR and PXR during ANIT-induced liver injury. Wild-type, FXR-null, and PXR-null mice were administered ANIT (75 mg/kg, po) and evaluated 48 h later for hepatotoxicity and messenger RNA (mRNA) expression of basolateral uptake (sodium taurocholate–cotransporting polypeptide, organic anion transporting polypeptide [Oatp] 1a1, Oatp1a4, Oatp1b2) and efflux transporters (organic solute transporter [Ost] , Ostβ, multidrug resistance–associated protein [Mrp] 3, Mrp4), as well as canalicular transporters (bile salt export pump [Bsep], Mrp2, multidrug resistance protein 2 [Mdr2], ATPase, class I, type 8B, member 1 [Atp8b1]). Livers from wild-type and PXR-null mice had comparable multifocal necrosis 48 h after ANIT. However, ANIT-treated FXR-null mice have fewer and smaller necrotic foci than wild-type mice but had scattered single-cell hepatocyte necrosis throughout the liver. Serum alanine transaminase, alkaline phosphatase (ALP), and direct bilirubin were increased in all genotypes, with higher ALP levels in FXR-null mice. Serum and liver unconjugated bile acids were higher in ANIT-treated FXR-null mice than the other two genotypes. ANIT induced mRNA expression of Mdr2, Bsep, and Atp8b1 in wild-type and PXR-null mice but failed to upregulate these genes in FXR-null mice. mRNA expression of uptake transporters declined in livers of all genotypes following ANIT treatment. ANIT increased Ostβ and Mrp3 mRNA in livers of wild-type and PXR-null mice but did not alter Ostβ mRNA in FXR-null mice. In conclusion, FXR deficiency enhances susceptibility of mice to ANIT-induced liver injury, likely a result of impaired induction of hepatobiliary efflux transporters and subsequent hepatic accumulation of unconjugated bile acids.

Resveratrol Inhibits Dioxin-Induced Expression of Human CYP1A1 and CYP1B1 by Inhibiting Recruitment of the Aryl Hydrocarbon Receptor Complex and RNA Polymerase II to the Regulatory Regions of the Corresponding Genes

Beedanagari, S. R., Bebenek, I., Bui, P., Hankinson, O. — 2009-06-15

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10µM resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes.

Discrimination of Tumorigenic Triazole Conazoles from Phenobarbital by Transcriptional Analyses of Mouse Liver Gene Expression

Nesnow, S., Ward, W., Moore, T., Ren, H., Hester, S. D. — 2009-06-15

Conazoles are fungicides used to control fungal growth in environmental settings and to treat humans with fungal infections. Mouse hepatotumorigenic conazoles display many of the same hepatic toxicologic responses as the mouse liver carcinogen phenobarbital (PB): constitutive androstane receptor (CAR) activation, hypertrophy, Cyp2b induction, and increased cell proliferation. The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Mice were administered diets containing PB (850 ppm), Pro (2500 ppm), or Tri (1800 ppm) for 4 and 30 days. Targeted transcriptomic analyses were conducted at the gene level examining differentially expressed genes (DEGs), and subsets of DEGs: cell cycle genes, and transcription factors. Analyses were also conducted on function, pathway and network levels examining Ingenuity Pathway Analysis Tox Lists and Canonical Pathways, and Gene-Go MetaCore dynamic networks and their central hubs. Genes expressed by PB or the two conazoles were also compared with those genes associated with human hepatocellular cancer. The results from these analyses indicated greater differences between PB and the two conazoles than similarities. Significant commonalities between the two conazole treatments were also noted. We posit that the transcriptional profiles of tissues exposed to toxic chemicals inherently contain their mechanisms of toxicity. We conclude that although PB and these 2 conazoles induce mouse liver tumors and exhibit similar toxicological responses, their transcriptional profiles are significantly different and thus their mechanisms of tumorigenic action are likely to differ.

Ochratoxin A-Mediated DNA and Protein Damage: Roles of Nitrosative and Oxidative Stresses

2009-06-15

Ochratoxin A (OTA) is a mycotoxin occurring in a variety of foods. OTA is nephrotoxic and nephrocarcinogenic in rodents. An OTA-mediated increase of the inducible nitric oxide synthase (iNOS) expression was observed in normal rat kidney renal cell line and in rat hepatocyte cultures, suggesting the induction of nitrosative stress. This was associated with an increased nuclear factor kappa-light chain enhancer of activated B cells activity. The potential consequences of iNOS induction were further investigated. A significant increase in the levels of protein nitrotyrosine residues was observed with OTA. In addition, OTA was found to increase the level of DNA abasic sites in both cell cultures system. This end point was used as an indirect measure of 8-nitroguanine formation. Treatment of the cells with L-N⁶-(1-iminoethyl) lysine, a specific inhibitor of iNOS activity, inhibited the OTA-mediated overnitration of proteins but did not reduce the level of DNA abasic sites. It was found previously that nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activators were able to restore the cellular defense against oxidative stress and could prevent DNA abasic sites in cell cultures. In the present study, pretreatment of the cells with activators of Nrf2 prevented OTA-mediated increase in lipid peroxidation, confirming the potential of Nrf2 activators to confer protection against OTA-mediated oxidative stress. In addition, it was found that Nrf2 activators could also prevent OTA-induced protein nitration and cytotoxicity. In conclusion, the present data further confirm oxidative stress as a key source of OTA-induced DNA damage and provide additional evidence for a role of this mechanism in OTA carcinogenicity. The exact role of nitrosative stress still remains to be established.

Effect of the Methoxychlor Metabolite HPTE on the Rat Ovarian Granulosa Cell Transcriptome In Vitro

Harvey, C. N., Esmail, M., Wang, Q., Brooks, A. I., Zachow, R., Uzumcu, M. — 2009-06-15

Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cyclic adenosine monophosphate (cAMP)-mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis and affects the messenger RNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10µM HPTE in the presence or absence of either 3 ng FSH/ml or 1mM cAMP for 48 h. Total RNA was isolated for real-time quantitative PCR and microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (~670 genes) than in cAMP-stimulated cells (~366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway and revealed that these effects include broader changes in gene expression.

Reproductive and Developmental Toxicity of a Pentabrominated Diphenyl Ether Mixture, DE-71, to Ranch Mink (Mustela vison) and Hazard Assessment for Wild Mink in the Great Lakes Region

2009-06-15

Polybrominated diphenyl ethers (PBDEs) are widespread persistent and bioaccumulative environmental contaminants. Recent scientific attention has focused on the developmental toxicity of PBDE commercial mixtures following perinatal exposure of rodents; however, these studies do not necessarily predict toxicity to highly exposed top predators, such as mink (Mustela vison). Here we assessed the effects of environmentally relevant doses (0, 0.1, 0.5, and 2.5 ppm [wt/wt] in feed) of a technical pentabrominated diphenyl ether mixture, DE-71, on reproductive performance of mink and on development of offspring exposed perinatally and post-weaning until 33 weeks. A dietary concentration that causes no effects on reproduction in rodents, 2.5-ppm DE-71, resulted in complete reproductive failure in these mink, while whelping rates were not affected at all lower does. Developmental effects in offspring were evident in 33-week-old juveniles, which were more sensitive to effects than their respective dams. Juvenile thyroid hormone homeostasis was also much more sensitive compared to rodents, and at 0.5-ppm DE-71, total triiodothyronine (T3) was significantly decreased in all males and females, even despite a compensatory increase of total thyroxine (T4) in females. T4-outer-ring deiodinase activity, mainly contributed by type II deiodinase, was not affected at any dose for any life stage, but thyroid follicular epithelium cell height was elevated in the 0.5-ppm–treated juveniles (p = 0.057). Ethoxyresorufin O-deethylase activity was significantly induced in all offspring at 33 weeks, most likely as a consequence of polybrominated dioxin, furan, or biphenyl impurities in DE-71. Biomonitoring of wild mink in the Great Lakes region indicated that most populations had lower concentrations than what are expected to affect thyroid hormone homeostasis, but margins of safety are small and mink around Hamilton Harbour exceeded the no observed adverse effect level for T3 disruption.

Relative Developmental Toxicity of Glycol Ether Alkoxy Acid Metabolites in the Embryonic Stem Cell Test as compared with the In Vivo Potency of their Parent Compounds

2009-06-15

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds.

Interference of Endocrine Disrupters with Thyroid Hormone Receptor-Dependent Transactivation

Hofmann, P. J., Schomburg, L., Kohrle, J. — 2009-06-15

Thyroid hormones regulate critical developmental processes and key metabolic pathways. A number of natural and synthetic substances have been identified which adversely interfere with the endocrine system. These so-called endocrine disrupters (ED) have mainly been studied for their impact on the gonadal hormone axis. The aim of this work was to develop a novel sensitive and convenient in vitro screening assay for the detection and characterization of potential ED of thyroid hormone (TH)–dependent transactivation of gene transcription and to apply this tool to test relevant environmental and nutritive ED compounds. We constructed a TH-responsive luciferase–based reporter plasmid and established a reporter gene assay in a 96 well microplate format using the human hepatocarcinoma cell line HepG2 as host system. Both the synthetic TH receptor (TR) agonist GC-1 and the antagonist NH-3 were used to evaluate the assay. Concentration-response data of test compounds (food constituents, isoflavones, ultraviolet-absorbers, pesticides, industrial chemicals) were recorded in activation assays. In addition, interference with TH-mediated transactivation was tested by coincubation of the ED with triiodothyronine (T₃) in competition assays. Most ED tested affected T₃ reporter gene activity at concentrations of 1µM or higher and displayed either agonistic or mixed agonistic/antagonistic activities. Effects of relevant ED occurred only at relatively high concentrations compared with the endogenous TR ligand T₃. However, on basis of their high production volumes and potential bioaccumulation of some fat-soluble ED our data indicate the need to carefully monitor certain ED for potential disruption of the TH system in intact organisms and humans.

Mechanisms of Quantum Dot Nanoparticle Cellular Uptake

Zhang, L. W., Monteiro-Riviere, N. A. — 2009-06-15

Due to the superior photoemission and photostability characteristics, quantum dots (QD) are novel tools in biological and medical applications. However, the toxicity and mechanism of QD uptake are poorly understood. QD nanoparticles with an emission wavelength of 655 nm are ellipsoid in shape and consist of a cadmium/selenide core with a zinc sulfide shell. We have shown that QD with a carboxylic acid surface coating were recognized by lipid rafts but not by clathrin or caveolae in human epidermal keratinocytes (HEKs). QD were internalized into early endosomes and then transferred to late endosomes or lysosomes. In addition, 24 endocytic interfering agents were used to investigate the mechanism by which QD enter cells. Our results showed that QD endocytic pathways are primarily regulated by the G-protein–coupled receptor associated pathway and low density lipoprotein receptor/scavenger receptor, whereas other endocytic interfering agents may play a role but with less of an inhibitory effect. Lastly, low toxicity of QD was shown with the 20nM dose in HEK at 48 h but not at 24 h by the live/dead cell assay. QD induced more actin filaments formation in the cytoplasm, which is different from the actin depolymerization by cadmium. These findings provide insight into the specific mechanism of QD nanoparticle uptake in cells. The surface coating, size, and charge of QD nanoparticles are important parameters in determining how nanoparticle uptake occurs in mammalian cells for cancer diagnosis and treatment, and drug delivery.

Estrogen and Tamoxifen Protect against Mn-Induced Toxicity in Rat Cortical Primary Cultures of Neurons and Astrocytes

Lee, E.-S. Y., Yin, Z., Milatovic, D., Jiang, H., Aschner, M. — 2009-06-15

Chronic exposure to manganese (Mn) leads to a neurological disorder, manganism, which shares multiple common features with idiopathic Parkinson disease (IPD). 17β-Estradiol (E2) and some selective estrogen receptor modulators, including tamoxifen (TX), afford neuroprotection in various experimental models of neurodegeneration. However, the neuroprotective effects and mechanisms of E2/TX in Mn-induced toxicity have yet to be documented. Herein, we studied the ability of E2/TX to protect rat cortical primary neuronal and astroglial cultures from Mn-induced toxicity. Cell viability, Western blot, and reactive oxygen species (ROS) generation were assessed. Results established that both E2 (10nM) and TX (1µM) attenuated Mn-induced toxicity. The protective effects of E2/TX were more pronounced in astrocytes versus neurons. The E2-mediated attenuation of Mn-induced ROS generation in astrocytes at 6-h treatment (where no cell death was detected) was mediated by a classical estrogen receptor (ER) pathway and the TX-mediated effect on Mn-induced ROS generation was not mediated via classical ER-dependent mechanisms and likely by its antioxidant properties. The phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway was involved in both E2- and TX-induced attenuation of Mn-induced ROS formation (6 h) in astrocytes. Treatments with Mn for a longer duration (24 h) led to significant cell death, and the protective effects of E2 and TX were (1) not mediated by a classical ER pathway and (2) associated with activation of both mitogen-activated protein kinase/extracellular signal-regulated kinase and PI3K/Akt signaling pathways. Taken together, the results suggest that both E2 and TX offer effective therapeutic means for neuroprotection against Mn-induced toxicity.

Neurotoxicogenomic Investigations to Assess Mechanisms of Action of the Munitions Constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus)

2009-06-15

Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (≤ 17), indicating that 2,6-DNT exposure had relatively little impact on the brain in comparison to RDX. Birds exhibiting RDX-induced seizures accumulated over 20x more RDX in brain tissues in comparison to non-seizing birds even within a common dose. In parallel, expression patterns were unrelated among seizing and non-seizing birds exposed to equivalent RDX doses. In birds experiencing seizures, genes related to neuronal electrophysiology and signal transduction were significantly affected. Comparative toxicology revealed strong similarity in acute exposure effects between RDX and the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) regarding both molecular mechanisms and putative mode of action. In a manner similar to DDT, we hypothesize that RDX elicits seizures by inhibition of neuronal cell repolarization postaction potential leading to heightened neuronal excitability and seizures facilitated by multiple molecular mechanisms.

Profiling the Reproductive Toxicity of Chemicals from Multigeneration Studies in the Toxicity Reference Database

2009-06-15

Multigeneration reproduction studies are used to characterize parental and offspring systemic toxicity, as well as reproductive toxicity of pesticides, industrial chemicals and pharmaceuticals. Results from 329 multigeneration studies on 316 chemicals have been digitized into standardized and structured toxicity data within the Toxicity Reference Database (ToxRefDB). An initial assessment of data quality and consistency was performed prior to profiling these environmental chemicals based on reproductive toxicity and associated toxicity endpoints. The pattern of toxicity across 75 effects for all 316 chemicals provided sets of chemicals with similar in vivo toxicity for future predictive modeling. Comparative analysis across the 329 studies identified chemicals with sensitive reproductive effects, based on comparisons to chronic and subchronic toxicity studies, as did the cross-generational comparisons within the multigeneration study. The general pattern of toxicity across all chemicals and the more focused comparative analyses identified 19 parental, offspring and reproductive effects with a high enough incidence to serve as targets for predictive modeling that will eventually serve as a chemical prioritization tool spanning reproductive toxicities. These toxicity endpoints included specific reproductive performance indices, male and female reproductive organ pathologies, offspring viability, growth and maturation, and parental systemic toxicities. Capturing this reproductive toxicity data in ToxRefDB supports ongoing retrospective analyses, test guideline revisions, and computational toxicology research.

Pulmonary Nanoparticle Exposure Disrupts Systemic Microvascular Nitric Oxide Signaling

2009-06-15

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO₂ (primary particle diameter ~1 µm) and TiO₂ nanoparticles (primary particle diameter ~21 nm) via aerosol inhalation at depositions of 4–90 µg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by ~60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.

Differential Hepatic Effects of Perfluorobutyrate Mediated by Mouse and Human PPAR-{alpha}

2009-06-15

Perfluorobutryate (PFBA) is a short chain perfluoroalkyl carboxylate that is structurally similar to perfluorooctanoate. Administration of PFBA can cause peroxisome proliferation, induction of peroxisomal fatty acid oxidation and hepatomegaly, suggesting that PFBA activates the nuclear receptor, peroxisome proliferator–activated receptor- (PPAR-). In this study, the role of PPAR- in mediating the effects of PFBA was examined using PPAR- null mice and a mouse line expressing the human PPAR- in the absence of mouse PPAR- (PPAR- humanized mice). PFBA caused upregulation of known PPAR- target genes that modulate lipid metabolism in wild-type and PPAR- humanized mice, and this effect was not found in PPAR- null mice. Increased liver weight and hepatocyte hypertrophy were also found in wild-type and humanized PPAR- mice treated with PFBA, but not in PPAR- null mice. Interestingly, hepatocyte focal necrosis with inflammatory cell infiltrate was only found in wild-type mice administered PFBA; this effect was markedly diminished in both PPAR- null and PPAR- humanized mice. Results from these studies demonstrate that PFBA can modulate gene expression and cause mild hepatomegaly and hepatocyte hypertrophy through a mechanism that requires PPAR- and that these effects do not exhibit a species difference. In contrast, the PPAR-–dependent increase in PFBA-induced hepatocyte focal necrosis with inflammatory cell infiltrate was mediated by the mouse PPAR- but not the human PPAR-. Collectively, these findings demonstrate that PFBA can activate both the mouse and human PPAR-, but there is a species difference in the hepatotoxic response to this chemical.

Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

Nemec, A. A., Barchowsky, A. — 2009-06-15

Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1 (HIF-1) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1 activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells.

Continuous Electrocardiogram Reveals Differences in the Short-Term Cardiotoxic Response of Wistar-Kyoto and Spontaneously Hypertensive Rats to Doxorubicin

Hazari, M. S., Haykal-Coates, N., Winsett, D. W., Costa, D. L., Farraj, A. K. — 2009-06-15

Electrocardiography (ECG) is one of the standard technologies used to monitor and assess cardiac function, and provide insight into the mechanisms driving myocardial pathology. Increased understanding of the effects of cardiovascular disease on rat ECG may help make ECG assessments in rat toxicology studies routine, thus facilitating continuous measurement of functional decrements associated with cardiotoxicant exposure. These studies seek to test the hypothesis that hypertensive rats are more susceptible to the short-term cardiotoxic effects of doxorubicin (DOX) when compared with normotensive rats with respect to continuously measured ECG endpoints. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats surgically implanted with radiotelemeters were treated once a week for three weeks with either vehicle, 1.25 (low), 2.5 (medium), or 5 (high) mg/kg DOX (i.p.). ECG, heart rate (HR), and core body temperature (T_co) were continuously monitored during the 1-week baseline and throughout the experimental period until rats were sacrificed 24 h after the third injection. DOX prevented normal body weight gain in both strains and significantly decreased diurnal HR and T_co of high DOX SH rats. In the ECG, SH rats had prolonged baseline PR intervals and QT_c when compared with WKY rats. All DOX-treated WKY rats subsequently developed PR interval prolongation; however only those treated with high DOX had increased QT_c. DOX caused an increase in ST interval in SH rats, and resulted in ECG morphology changes. The number of arrhythmias due to DOX was increased in both strains. In conclusion, ECG analysis can reveal underlying cardiovascular disease as a risk factor in the heart's response to toxicant-induced injury in the rat; and be a valuable tool to evaluate baseline vulnerability and assess cardiotoxicity.

Population-Based Discovery of Toxicogenomics Biomarkers for Hepatotoxicity Using a Laboratory Strain Diversity Panel

Harrill, A. H., Ross, P. K., Gatti, D. M., Threadgill, D. W., Rusyn, I. — 2009-06-15

Toxicogenomic studies are increasingly used to uncover potential biomarkers of adverse health events, enrich chemical risk assessment, and to facilitate proper identification and treatment of persons susceptible to toxicity. Current approaches to biomarker discovery through gene expression profiling usually utilize a single or few strains of rodents, limiting the ability to detect biomarkers that may represent the wide range of toxicity responses typically observed in genetically heterogeneous human populations. To enhance the utility of animal models to detect response biomarkers for genetically diverse populations, we used a laboratory mouse strain diversity panel. Specifically, mice from 36 inbred strains derived from Mus mus musculus, Mus mus castaneous, and Mus mus domesticus origins were treated with a model hepatotoxic agent, acetaminophen (300 mg/kg, ig). Gene expression profiling was performed on liver tissue collected at 24 h after dosing. We identified 26 population-wide biomarkers of response to acetaminophen hepatotoxicity in which the changes in gene expression were significant across treatment and liver necrosis score but not significant for individual mouse strains. Importantly, most of these biomarker genes are part of the intracellular signaling involved in hepatocyte death and include genes previously associated with acetaminophen-induced hepatotoxicity, such as cyclin-dependent kinase inhibitor 1A (p21) and interleukin 6 signal transducer (Il6st), and genes not previously associated with acetaminophen, such as oncostatin M receptor (Osmr) and MLX interacting protein like (Mlxipl). Our data demonstrate that a multistrain approach may provide utility for understanding genotype-independent toxicity responses and facilitate identification of novel targets of therapeutic intervention.

Nervous and Vestibular Toxicities of Acrylonitrile and Iminodipropionitrile

Llorens, J., Soler-Martin, C., Cutillas, B., Saldana-Ruiz, S. — 2009-06-15

On the Mechanism of Nitriles Toxicity

Khan, H. A., Alhomida, A. S., Arif, I. A. — 2009-06-15

ERRATUM

2009-06-15

Potential Clinical Significance of EGFR-Mediated Signaling following Inorganic Arsenic Exposure in Human Lung

Eblin, K. — 2009-05-18

Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays

2009-05-18

With the advent of new technologies (e.g., genomics, automated analyses, and in vivo monitoring), new regulations (e.g., the reduction of animal tests by the European REACH), and new approaches to toxicology (e.g., Toxicity Testing in the 21st Century, National Research Council), the field of regulatory genetic toxicology is undergoing a serious re-examination. Within this context, Toxicological Sciences has published a series of articles in its Forum Section on the theme, "Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation" (beginning with Goodman et al.). As a contribution to the Forum discussions, we present current methods for evaluating mutagenic/genotoxic risk using standard genotoxicity test batteries, and suggest ways to address and incorporate new technologies. We recognize that the occurrence of positive results in relation to cancer prediction has led to criticism of in vitro mammalian cell genetic toxicity assays. We address criticism of test results related to weak positives, associated only with considerable toxicity, only seen at high concentrations, not accompanied by positive results in the other tests of standard test batteries, and/or not correlating well with rodent carcinogenicity tests. We suggest that the problems pointed out by others with these assays already have been resolved, to a large extent, by international groups working to update assay protocols, and by changes in data interpretation at regulatory agencies. New guidances at the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration improve data evaluation and help refocus risk assessment. We discuss the results of international groups working together to integrate new technologies and evaluate new tests, including human monitoring. We suggest that strategies for identifying human health risks should naturally change to integrate new technologies; however, changes should be made only when justified by strong scientific evidence of improvement in the risk assessment paradigm.

A Computational Model of the Hypothalamic-Pituitary-Gonadal Axis in Male Fathead Minnows Exposed to 17{alpha}-Ethinylestradiol and 17{beta}-Estradiol

2009-05-18

Estrogenic chemicals in the aquatic environment have been shown to cause a variety of reproductive anomalies in fish including full sex reversal, intersex, and altered population sex ratios. Two estrogens found in the aquatic environment, 17-ethinylestradiol (EE₂) and 17β-estradiol (E₂), have been measured in wastewater treatment effluents and have been shown to cause adverse effects in fish. To further our understanding of how estrogen exposure affects reproductive endpoints in the male fathead minnow (FHM, Pimephales promelas), a physiologically based computational model was developed of the hypothalamic-pituitary-gonadal (HPG) axis. Apical reproductive endpoints in the model include plasma steroid hormone and vitellogenin concentrations. Using Markov chain Monte Carlo simulation, the model was calibrated with data from unexposed FHM, and FHM exposed to EE₂ and E₂. Independent experimental data sets were used to evaluate model predictions. We found good agreement between our model predictions and a variety of measured reproductive endpoints, although the model underpredicts unexposed FHM reproductive endpoint variances, and overpredicts variances in estrogen-exposed FHM. We conclude that this model provides a robust representation of the HPG axis in male FHM.

Phenobarbital Elicits Unique, Early Changes in the Expression of Hepatic Genes that Affect Critical Pathways in Tumor-Prone B6C3F1 Mice

Phillips, J. M., Burgoon, L. D., Goodman, J. I. — 2009-05-18

At 2 and 4 weeks following treatment with phenobarbital (PB), the classical nongenotoxic rodent liver carcinogen, we elucidated unique gene expression changes (both induction and repression) in liver tumor-susceptible B6C3F1 mice, as compared with the relatively resistant C57BL/6. Based on their cancer-related roles, we believe that altered expression of at least some of these genes might underlie PB-induced liver tumorigenesis. Putative constitutive active/androstane (CAR) response elements (CAREs), a subset of PB response elements, were present within multiple genes whose expression was uniquely altered in the B6C3F1 mice, suggesting a role for CAR in their regulation. Additionally, three DNA methyltransferase genes (Dnmt1, Dnmt3a, and Dnmt3b) were repressed uniquely in the tumor-prone B6C3F1 mice, and all possess putative CAREs, providing a potential direct link between PB and expression of key genes that regulate DNA methylation status. Previously, we demonstrated that PB-elicited unique regions of altered methylation (RAMs) in B6C3F1 mice, as compared with the relatively resistant C57BL/6, at 2 and 4 weeks, and annotation of the regions harboring these changes revealed 51 genes. This is extended by the current study, which employed RNA isolated from the same liver tissue used in the earlier investigations. Genes elucidated from both the methylation and expression analyses are involved in identical processes/pathways (e.g., cell cycle, apoptosis, angiogenesis, epithelial-mesenchymal cell transition, invasion/metastasis, and mitogen-activated protein kinase, transforming growth factor-beta, and Wnt signaling). Therefore, these changes might represent very early events that directly contribute to PB-induced tumorigenesis. It is instructive to consider the possibility that, in a hypothesis-driven fashion, these genes are initial candidates that could be utilized to develop a biomarker "fingerprint" of early exposure to PB and PB-like compounds.

Competitive Binding of Poly- and Perfluorinated Compounds to the Thyroid Hormone Transport Protein Transthyretin

2009-05-18

Due to their unique surfactant properties, poly- and perfluorinated compounds (PFCs) have been extensively used and can be found all over the environment. Concern about their environmental fate and toxicological properties has initiated several research projects. In the present study, we investigated if PFCs can compete with thyroxine (T₄, i.e., the transport form of thyroid hormone) for binding to the human thyroid hormone transport protein transthyretin (TTR). Such competitive capacity may lead to decreased thyroid hormone levels as previously reported for animals exposed to PFCs. Twenty-four PFCs, together with 6 structurally similar natural fatty acids, were tested for binding capacity in a radioligand-binding assay. The binding potency decreased in the order: perfluorohexane sulfonate > perfluorooctane sulfonate/perfluorooctanoic acid > perfluoroheptanoic acid > sodium perfluoro-1-octanesulfinate > perfluorononanoic acid, with TTR binding potencies 12.5–50 times lower than the natural ligand T₄. Some lower molecular weight compounds with structural similarity to these PFCs were > 100 times less potent than T₄. Simple descriptors based on the two-dimensional molecular structures of the compounds were used to visualize the chemical variation and to model the structure-activity relationship for the competitive potencies of the TTR-binding compounds. The models indicated the dependence on molecular size and functional groups but demanded a more detailed description of the chemical properties and data for validation and further quantitative structure-activity relationship (QSAR) development. Competitive binding of PFCs to TTR, as observed for human TTR in the present study, may explain altered thyroid hormone levels described for PFC-exposed rats and monkeys. Median human blood levels of the most potent TTR-binding PFCs are one to two orders of magnitude lower than concentration at 50% inhibition (IC₅₀) values determined in the present study. In addition, this study contributes to the understanding of the bioaccumulation of PFCs in man and possibly in other wildlife species.

Antioxidant Responses and NRF2 in Synergistic Developmental Toxicity of PAHs in Zebrafish

Timme-Laragy, A. R., Van Tiem, L. A., Linney, E. A., Di Giulio, R. T. — 2009-05-18

Early piscine life stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with coexposure to the aryl hydrocarbon receptor agonist β-naphthoflavone (BNF) and cytochrome P4501A inhibitor -naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF + ANF combination, and a prooxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione (GSH) and attempted to modulate deformities using the GSH synthesis inhibitor L-buthionine (S,R)-sulfoximine (BSO) and increase GSH pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF + ANF coexposure significantly increased expressions of superoxide dismutase 1 and 2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some GSH levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF + ANF exposures, and exacerbated BNF + ANF–related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity and that NRF2 is protective against prooxidant and PAH challenges during development.

Cloning, Expression, and Biochemical Properties of CPOX4, a Genetic Variant of Coproporphyrinogen Oxidase that Affects Susceptibilitly to Mercury Toxicity in Humans

Li, T., Woods, J. S. — 2009-05-18

Coproporphyrinogen oxidase (CPOX) catalyzes the two-step decarboxylation of coproporphyrinogen-III to protoporphyrinogen-IX in the heme biosynthetic pathway. Previously we described a specific polymorphism (A814C) in exon 4 of the human CPOX gene (CPOX4) and demonstrated that CPOX4 is associated with both modified urinary porphyrin excretion and increased neurobehavioral deficits among human subjects with low-level mercury (Hg) exposure. Here, we sought to characterize the gene products of CPOX and CPOX4 with respect to biochemical and kinetic properties. Coproporphyrinogen-III was incubated with recombinantly expressed and purified human CPOX and CPOX4 enzymes at various substrate concentrations, with or without Hg²⁺ present. Both CPOX and CPOX4 formed protoporphyrinogen-IX from coproporphyrinogen-III; however, the affinity of CPOX4 was twofold lower than that of CPOX (CPOX K_m = 0.30µM, V_max = 0.52 pmol protoporphyrin-IX; CPOX4 K_m = 0.54µM, V_max = 0.33 pmol protoporphyrin-IX). Hg²⁺ specifically inhibited the second step of coproporphyrinogen-III decarboxylation (harderoporphyrinogen to protoporphyrinogen-IX) in a dose dependent manner. We also compared the catalytic activities of CPOX and CPOX4 in human liver samples. The specific activities of CPOX in mutant livers were significantly lower (40–50%) than those of either wild-type or heterozygous. Additionally, enzymes from mutant, heterozygous and wild-type livers were comparably inhibited by Hg²⁺ (10µM), decreasing CPOX4 activity to 25% that of the wild-type enzyme. These findings suggest that CPOX4 may predispose to impaired heme biosynthesis, which is limited further by Hg exposure. These effects may underlie increased susceptibility to neurological deficits previously observed in Hg-exposed humans with CPOX4.

Oxidative Stress and Sodium Methyldithiocarbamate-Induced Modulation of the Macrophage Response to Lipopolysaccharide In Vivo

Pruett, S. B., Cheng, B., Fan, R., Tan, W., Sebastian, T. — 2009-05-18

Sodium methyldithiocarbamate (SMD) is the third most abundantly used conventional pesticide in the United States, and hundreds of thousands of persons are exposed to this compound or its major breakdown product, methylisothiocyanate, at levels greater than recommended by the Environmental Protection Agency. A previous study suggests three mechanisms of action involved to some degree in the inhibition of inflammation and decreased resistance to infection caused by exposure of mice to the compound. One of these mechanisms is oxidative stress. The purpose of the present study was to confirm that this mechanism is involved in the effects of SMD on cytokine production by peritoneal macrophages and to further characterize its role in altered cytokine production. Results indicated that SMD significantly decreased the intracellular concentration of reduced glutathione (GSH), suggesting oxidative stress. This was further indicated by the upregulation of genes involved in the "response to oxidative stress" as determined by microarray analysis. These effects were associated with the inhibition of lipopolysaccharide (LPS)-induced production of several proinflammatory cytokines. Experimental depletion of GSH with buthionine sulfoximine (BSO) partially prevented the decrease in LPS-induced interleukin (IL)-6 production caused by SMD and completely prevented the decrease in IL-12. In contrast, BSO plus SMD substantially enhanced the production of IL-10. These results along with results from a previous study are consistent with the hypothesis that SMD causes oxidative stress, which contributes to modulation of cytokine production. However, oxidative stress alone cannot explain the increased IL-10 production caused by SMD.

Role of GRP78/BiP Degradation and ER Stress in Deoxynivalenol-Induced Interleukin-6 Upregulation in the Macrophage

Shi, Y., Porter, K., Parameswaran, N., Bae, H. K., Pestka, J. J. — 2009-05-18

The trichothecene mycotoxin deoxynivalenol (DON) induces systemic expression of the interleukin-6 (IL-6) and other proinflammatory cytokines in the mouse. The purpose of this study was to test the hypothesis that DON triggers an endoplasmic reticulum (ER) stress response in murine macrophages capable of driving IL-6 gene expression. DON at concentrations up 5000 ng/ml. was not cytotoxic to peritoneal cells. However, DON markedly decreased protein levels but not the mRNA levels of glucose-regulated protein (GRP) 78 (BiP), a chaperone known to mediate ER stress. Inhibitor studies suggested that DON-induced GRP78 degradation was cathepsin and calpain dependent but was proteosome-independent. RNAi-mediated knockdown of GRP78 resulted in increased IL-6 gene expression indicating a potential downregulatory role for this chaperone. GRP78 is critical to the regulation of the two transcription factors, X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), which bind to cAMP-response element (CRE) and drive expression of CRE-dependent genes such as IL-6. DON exposure was found to increase IRE1 protein, its modified products spliced XBP1 mRNA and XBP1 protein as well as ATF6. Knockdown of ATF6 but not XBP1 partially inhibited DON-induced IL-6 expression in the macrophages. Three other trichothecenes (satratoxin G, roridin, T-2 toxin) and the ribosome inhibitory protein ricin were also found to induce GRP78 degradation suggesting that other translation inhibitors might evoke ER stress. Taken together, these data suggest that in the macrophage DON induces GRP78 degradation and evokes an ER stress response that could contribute, in part, to DON-induced IL-6 gene expression.

Is Toluene Diamine a Sensitizer and is there Cross-Reactivity between Toluene Diamine and Toluene Diisocyanate?

Vanoirbeek, J. A. J., De Vooght, V., Synhaeve, N., Nemery, B., Hoet, P. H. M. — 2009-05-18

Toluene diamine (TDA) is formed when toluene diisocyanate (TDI), a potent sensitizer, comes in contact with an aqueous environment. The sensitizing capacity of TDA and the cross-reactivity between TDI and TDA are unknown. TDA (5–25%) and TDI (0.3%), dissolved in acetone/olive oil (AOO) (4:1) were tested in the mouse local lymph node assay (LLNA). To determine the capacity of TDA to elicit an asthmatic response and to determine the cross-reaction with TDI, a locally developed experimental mouse model of chemical-induced asthma was used. On days 1 and 8, BALB/c mice received 20 µl of TDI (0.3%), TDA (20%), or AOO (4:1) on each ear. On day 15, they received an intranasal instillation of TDI (0.1%), TDA (0.5%) or AOO (3:2). The EC₃ of TDA in the LLNA is 19%. In the model of chemical-induced asthma, TDI induced a ventilatory response [increased Penh after challenge; increased airway hyperreactivity (AHR)], inflammatory changes (bronchoalveolar lavage neutrophils), and immunological changes (increased CD19⁺ lymphocytes, IL-4 and total serum IgE), whereas TDA did not show any of these responses. Mice sensitized with TDI and challenged with TDA also did not show any airway or inflammatory response, although they had increased levels of total serum IgE. Mice sensitized with TDA and challenged with TDI did not show any response. According to the classification of sensitizers in the LLNA, TDA is a weak dermal sensitizer. In the experimental mouse model of chemical-induced asthma, TDA does not act as a respiratory sensitizer, at the concentration used. No cross-reactivity between TDI and TDA was found.

Patterns of Immunotoxicity Associated with Chronic as Compared with Acute Exposure to Chemical or Physical Stressors and their Relevance with Regard to the Role of Stress and with Regard to Immunotoxicity Testing

Pruett, S. B., Fan, R., Zheng, Q., Schwab, C. — 2009-05-18

Previous studies have demonstrated that the stress response induced by some drugs and chemicals contributes in a predictable way to alteration of particular immunological parameters in mice. It has not been determined if mice can become tolerant or habituated with regard to the stress response and consequent immunological effects. Addressing this issue was the purpose of the present study. Mice were dosed daily for 28 days with atrazine, ethanol, propanil, or subjected to restraint, which are known to induce neuroendocrine stress responses and thereby to alter several immunological parameters. On day 29, a blood sample was taken and the spleen was removed for analysis of cellular phenotypes, differential cell counts (for blood), and natural killer (NK) cell activity. Corticosterone concentration at various times after dosing (or restraint) was also measured. Comparison of these results with results from previous studies with a single acute exposure revealed that the corticosterone response was almost completely absent in mice treated with ethanol, reduced in mice treated with restraint and propanil, and for atrazine the response was the same as noted for acute exposure. In most cases, the changes in immunological parameters were consistent with expectations based on these corticosterone responses. However, in a few cases (e.g., NK cell activity), it was clear that there were effects not mediated by stress. These results indicate that the nature of the stressor determines whether mice become tolerant with regard to the stress response and consequent immunological effects. This finding has practical implications for safety testing in mice.

The Homeostasis of Phosphatidylcholine and Lysophosphatidylcholine in Nervous Tissues of Mice was not Disrupted after Administration of Tri-o-cresyl Phosphate

Hou, W.-Y., Long, D.-X., Wu, Y.-J. — 2009-05-18

Neuropathy target esterase (NTE) is proven to act as a lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. In sensitive species, organophosphate (OP)–induced delayed neurotoxicity is initiated when NTE is inhibited by > 70% and then aged. It is hypothesized that homeostasis of phosphatidylcholine (PC) and/or lysophosphatidylcholine (LPC) in mice might be disrupted by the OPs since NTE and other phospholipases could be inhibited. To test this hypothesis, we treated mice using tri-o-cresyl phosphate (TOCP), which can inhibit and age NTE. Phenylmethylsulfonyl fluoride (PMSF), which inhibits NTE but cannot age, was used as a negative control. Effects on activity of NTE, LysoPLA, and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE in the brain, spinal cord, and sciatic nerve were examined. The results showed that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB, and PLB were significantly inhibited in both TOCP- and PMSF-treated mice, and the inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient. The NTE inhibited by TOCP was of the aged type, while nearly all NTE inhibited by PMSF was of the unaged type. Although the GPC level was remarkedly decreased, no significant change of PC and LPC levels was observed. However, the inhibition of these enzymes in mice by TOCP exhibited different characteristics from the TOCP-treated hens that we previously reported, which indicates that these enzymes were inhibited and then recovered more rapidly in mice than in hens. All results suggest that PC and LPC homeostasis was not disrupted in mice after exposure to TOCP. Differences in inhibition of NTE, LysoPLA, and PLB activities by TOCP between mice and hens may elucidate why these two species display different signs after exposure to the same neuropathic OPs.

Selective Brain Uptake and Behavioral Effects of the Cyanobacterial Toxin BMAA ({beta}-N-Methylamino-L-alanine) following Neonatal Administration to Rodents

Karlsson, O., Lindquist, N. G., Brittebo, E. B., Roman, E. — 2009-05-18

Cyanobacteria are extensively distributed in terrestrial and aquatic environments all over the world. Most cyanobacteria can produce the neurotoxin β-N-methylamino-L-alanine (BMAA), which has been detected in several water systems and could accumulate in food chains. The aim of the study was to investigate the transfer of BMAA to fetal and neonatal brains and the effects of BMAA on the development of behavioral characteristics during the brain growth spurt (BGS) in rodents. Pregnant and neonatal mice were given an injection of ³H-BMAA on gestational day 14 and postnatal day (PND) 10, respectively, and processed for tape-section autoradiography. The study revealed transplacental transfer of ³H-BMAA and a significant uptake in fetal mouse brain. The radioactivity was specifically located in the hippocampus, striatum, brainstem, spinal cord and cerebellum of 10-day-old mice. The effect of repeated BMAA treatment (200 or 600 mg/kg sc) during BGS on rat behavior was also studied. BMAA treatment on PND 9–10 induced acute alterations, such as impaired locomotor ability and hyperactivity, in the behavior of neonatal rats. Furthermore, rats given the high dose of BMAA failed to habituate to the test environment when tested at juvenile age. In conclusion, the results demonstrated that BMAA was transferred to the neonatal brain and induced significant changes in the behavior of neonatal rats following administration during BGS. The observed behavioral changes suggest possible cognitive impairment. Increased information on the long-term effects of BMAA on cognitive function following fetal and neonatal exposure is required for assessment of the risk to children's health.

Neurobehavioral Effects of Acute Exposure to Four Solvents: Meta-analyses

Benignus, V. A., Bushnell, P. J., Boyes, W. K., Eklund, C., Kenyon, E. M. — 2009-05-18

Meta- and reanalyses of the available data for the neurobehavioral effects of acute inhalation exposure to toluene were reported by Benignus et al. The present study was designed to test the generality of the toluene results in as many other solvents as possible by further meta- and reanalyses. Sufficient data for meta-analyses were found for only four solvents; toluene, trichloroethylene, perchloroethylene, and 1,1,1-trichloroethane. The results for these solvents showed that rats were less affected by each of the solvents when they were tested in highly motivating situations, for example, rewarded for rapid or correct responding or escape from electrical shock, compared with less motivating circumstances. The four solvents did not differ significantly in potency on any outcome measure when dose was expressed as molar brain concentration. When tested in tasks with low-motivational contingencies, the dose-effect curves of humans (reaction times) and rats (electrophysiological responses to visual stimuli) were not significantly different. However, on an exploratory follow-up analysis, humans were less sensitive than rats. No human data were found to test whether species differed under strong motivation. Dose-equivalence curves were derived for extrapolating to human effects from rat data.

Exposure to Polybrominated Diphenyl Ethers 203 and 206 during the Neonatal Brain Growth Spurt Affects Proteins Important for Normal Neurodevelopment in Mice

Viberg, H. — 2009-05-18

The period of rapid brain growth and development (BGS) is postnatal in mice and rats, spanning the first 3–4 weeks of life, reaching its peak around postnatal day 10, whereas in humans, the BGS is perinatal. CaMKII, GAP-43, synaptophysin, and tau play important roles during the BGS. One class of flame retardants, polybrominated diphenyl ethers (PBDEs), is present and increasing in the environment and in human milk. The only congener still in use, decabrominated diphenyl ether (PBDE 209), is thought to be debrominated into lower brominated congeners. In the present study, nona- and octabrominated PBDEs were examined. Neonatal mice were exposed to 21 µmol PBDE 203 or 206/kg bodyweight on postnatal day 10, and different brain regions were analyzed for CaMKII, GAP-43, synaptophysin, and tau, 24 h after exposure. The protein analysis showed that CaMKII and synaptophysin increased significantly in the hippocampus, but not in the cerebral cortex, after neonatal exposure to PBDE 203 or 206. Furthermore, there were no significant changes in the levels of GAP-43 and tau in the cerebral cortex or hippocampus after neonatal exposure to PBDE 203 or 206. This shows that PBDE 203 and 206 affect important proteins involved in normal maturation of the brain and strengthens our findings that highly brominated PBDEs cause developmental neurotoxicity. In addition, the increases in CaMKII and synaptophysin are the same changes seen after neonatal PBDE 209 exposure; supporting the suggestion that PBDE 209 must be metabolized, likely debrominated into lower brominated PBDEs, to exert its neurotoxic effects.

Pulmonary Evaluation of Permissible Exposure Limit of Syntroleum S-8 Synthetic Jet Fuel in Mice

Wong, S. S., Thomas, A., Barbaris, B., Lantz, R. C., Witten, M. L. — 2009-05-18

No current studies have systematically examined pulmonary health effects associated with Syntroleum S-8 synthetic jet fuel (S-8). In order to gain an understanding about the threshold concentration in which lung injury is observed, C57BL/6 male mice were nose-only exposed to S-8 for 1 h/day for 7 days at average concentrations of 0 (control), 93, 352, and 616 mg/m³. Evaluation of pulmonary function, airway epithelial barrier integrity, and pathohistology was performed 24 h after the final exposures. Significant decreases were detected in expiratory lung resistance and total lung compliance of the 352 mg/m³ group, for which no clear concentration-dependent alterations could be determined. No significant changes in respiratory permeability were exhibited, indicating that there was no loss of epithelial barrier integrity following S-8 exposure. However, morphological examination and morphometric analysis of distal lung tissue, by using transmission electron microscopy, revealed cellular damage in alveolar type II epithelial cells, with significant increases in volume density of lamellar bodies/vacuoles at 352 and 616 S-8 mg/m³. Moreover, terminal bronchiolar Clara injury, as evidenced by apical membrane blebs, was observed at relatively low concentrations, suggesting if this synthetic jet fuel is utilized, the current permissible exposure limit of 350 mg/m³ for hydrocarbon fuels should cautiously be applied.

Physiologically Based Pharmacokinetic Model of Methyl Tertiary Butyl Ether and Tertiary Butyl Alcohol Dosimetry in Male Rats Based on Binding to {alpha}2u-Globulin

Leavens, T. L., Borghoff, S. J. — 2009-05-18

Current physiologically based pharmacokinetic (PBPK) models for the fuel additive methyl tertiary butyl ether (MTBE) and its metabolite tertiary butyl alcohol (TBA) have not included a mechanism for chemical binding to the male rat–specific protein 2u-globulin, which has been postulated to be responsible for renal effects in male rats observed in toxicity and carcinogenicity studies with MTBE. The objective of this work was to expand the previously published models for MTBE to include binding to 2u-globulin in the kidney of male rats. In the model, metabolism of MTBE was assumed to occur only in the liver via two saturable pathways. TBA metabolism was assumed to occur only in the liver via one saturable, low-affinity pathway and to be inducible following repeated exposures. The binding of MTBE and TBA to 2u-globulin was modeled as saturable and competitive and was assumed to only affect the rate of hydrolysis of 2u-globulin in the kidney. The developed model characterized the differences in kidney concentrations of MTBE and TBA in male versus female rats from inhalation exposures to MTBE, as well as the observed changes in blood and tissue concentrations from repeated exposure to TBA. The model-predicted binding affinity of MTBE to 2u-globulin was greater than TBA, and the hydrolysis rate of chemically bound 2u-globulin was approximately 30% of the unbound protein. This PBPK model supports the role of MTBE and TBA binding to the male rat–specific protein 2u-globulin as essential for predicting concentrations of these chemicals in the kidney following exposure.

Comparative Analysis of Novel Noninvasive Renal Biomarkers and Metabonomic Changes in a Rat Model of Gentamicin Nephrotoxicity

2009-05-18

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined ¹H-NMR and gas chromatography–mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, sc) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of ¹H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S₁ and S₂ proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.

Arsenic Activates EGFR Pathway Signaling in the Lung

Andrew, A. S., Mason, R. A., Memoli, V., Duell, E. J. — 2009-05-18

Arsenic is an established lung carcinogen, however, the carcinogenic mechanisms are currently under investigation. Phosphorylation of the epidermal growth factor receptor (EGFR) has been reported with arsenic exposure in bladder cells. EGFR is a tyrosine kinase transmembrane receptor that regulates important processes in carcinogenesis, including cell survival, cell cycle progression, tumor invasion, and angiogenesis. We investigated the mechanisms of EGFR pathway activation by levels of arsenic relevant to human exposure scenarios both in vitro using cultured lung epithelial cells, and in lung tumors samples from New England Lung Cancer Study participants. Toenail arsenic levels were used as an internal biomarker of arsenic exposure. Our in vitro data suggest that arsenic increases levels of the EGFR ligand, heparin binding-EGF, and activate EGFR phosphorylation in the lung. Downstream of EGFR, arsenic exposure increased pERK and cyclin D1 levels. These effects were inhibited by treatment of cultured cells with the EGFR tyrosine kinase inhibitor, Tarceva (erlotinib). In a consecutive series of human lung tumor specimens, pEGFR protein levels were higher in subjects with elevated toenail arsenic levels compared to those with low exposure (odds ratio adjusted for other factors, OR 4.1 (95% confidence interval 1.1–15.6) (p = 0.04). These data suggest that arsenic exposure may stimulate EGFR pathway activation in the lung. Moreover, the tumors that arise in arsenic-exposed individuals also exhibit signs of EGFR pathway dysregulation. Further work is needed to assess the clinical utility of targeting the EGFR pathway in subgroups of lung cancer patients who have been exposed to elevated levels of arsenic.

Toward a Public Toxicogenomics Capability for Supporting Predictive Toxicology: Survey of Current Resources and Chemical Indexing of Experiments in GEO and ArrayExpress

Williams-Devane, C. R., Wolf, M. A., Richard, A. M. — 2009-05-18

A publicly available toxicogenomics capability for supporting predictive toxicology and meta-analysis depends on availability of gene expression data for chemical treatment scenarios, the ability to locate and aggregate such information by chemical, and broad data coverage within chemical, genomics, and toxicological information domains. This capability also depends on common genomics standards, protocol description, and functional linkages of diverse public Internet data resources. We present a survey of public genomics resources from these vantage points and conclude that, despite progress in many areas, the current state of the majority of public microarray databases is inadequate for supporting these objectives, particularly with regard to chemical indexing. To begin to address these inadequacies, we focus chemical annotation efforts on experimental content contained in the two primary public genomic resources: ArrayExpress and Gene Expression Omnibus. Automated scripts and extensive manual review were employed to transform free-text experiment descriptions into a standardized, chemically indexed inventory of experiments in both resources. These files, which include top-level summary annotations, allow for identification of current chemical-associated experimental content, as well as chemical-exposure–related (or "Treatment") content of greatest potential value to toxicogenomics investigation. With these chemical-index files, it is possible for the first time to assess the breadth and overlap of chemical study space represented in these databases, and to begin to assess the sufficiency of data with shared protocols for chemical similarity inferences. Chemical indexing of public genomics databases is a first important step toward integrating chemical, toxicological and genomics data into predictive toxicology.

High Doses of Intravenously Administered Titanium Dioxide Nanoparticles Accumulate in the Kidneys of Rainbow Trout but with no Observable Impairment of Renal Function

2009-05-18

Our recent work suggests limited uptake of unstabilized metal oxide nanoparticles via water into fish, however, some other studies have indicated such exposures can induce oxidative stress. To investigate tissue distribution and toxicity of titanium dioxide (TiO₂) nanoparticles that may enter into fish, we conducted a series of injection studies. Rainbow trout (Oncorhynchus mykiss) were intravenously injected with 100 µg TiO₂ nanoparticles and the content of titanium in blood, brain, gills, liver, and kidney quantified at time points between 6 h and 90 days using inductively coupled plasma optical emission spectroscopy. Injected Ti was concentrated in the kidneys and remained there up to 21 days, however, there was evidence of clearance of TiO₂ at 90 days. Ti accumulation in the liver was 15 times lower than in the kidney with no apparent clearance. Using TEM we showed nanoparticles were localized in tissue vesicles surrounding the kidney tubules. In a second injection study, rainbow trout were injected with 100 µg TiO₂ and plasma samples from individual fish analyzed for total protein and creatinine content at time points between 6 h and 21 days to assess for possible effects on kidney function. No effect of TiO₂ on total plasma protein content or creatinine concentrations were found indicating that neither urine production nor glomerular filtration rate were affected. We conclude that in trout upon a single high dose exposure of TiO₂ nanoparticles via the bloodstream, TiO₂ accumulates in the kidneys but has minimal effect on kidney function.

Protection against Aflatoxin B1 in Rat--A New Look at the Link between Toxicity, Carcinogenicity, and Metabolism

Ellis, E. M. — 2009-04-30

Probabilistic Exposure Analysis for Chemical Risk Characterization

Bogen, K. T., Cullen, A. C., Frey, H. C., Price, P. S. — 2009-04-30

This paper summarizes the state of the science of probabilistic exposure assessment (PEA) as applied to chemical risk characterization. Current probabilistic risk analysis methods applied to PEA are reviewed. PEA within the context of risk-based decision making is discussed, including probabilistic treatment of related uncertainty, interindividual heterogeneity, and other sources of variability. Key examples of recent experience gained in assessing human exposures to chemicals in the environment, and other applications to chemical risk characterization and assessment, are presented. It is concluded that, although improvements continue to be made, existing methods suffice for effective application of PEA to support quantitative analyses of the risk of chemically induced toxicity that play an increasing role in key decision-making objectives involving health protection, triage, civil justice, and criminal justice. Different types of information required to apply PEA to these different decision contexts are identified, and specific PEA methods are highlighted that are best suited to exposure assessment in these separate contexts.

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High Doses of Intravenously Administered Titanium Dioxide Nanoparticles Accumulate in the Kidneys of Rainbow Trout but with no Observable Impairment of Renal Function

Protection against Aflatoxin B1 in Rat--A New Look at the Link between Toxicity, Carcinogenicity, and Metabolism

Probabilistic Exposure Analysis for Chemical Risk Characterization