Toxicological Sciences - recent issues

Fri, 13 Nov 2009 13:24:05 PST

COVER

Fri, 13 Nov 2009 13:24:05 PST

EDITORIAL BOARD

Fri, 13 Nov 2009 13:24:06 PST

SUBSCRIPTIONS

Fri, 13 Nov 2009 13:24:06 PST

Long-term Inhalation Toxicity Studies with Multiwalled Carbon Nanotubes: Closing the Gaps or Initiating the Debate?

Warheit, D. B. — Fri, 13 Nov 2009 13:24:06 PST

State of Academic Knowledge on Toxicity and Biological Fate of Quantum Dots

Pelley, J. L., Daar, A. S., Saner, M. A. — Fri, 13 Nov 2009 13:24:06 PST

Quantum dots (QDs), an important class of emerging nanomaterial, are widely anticipated to find application in many consumer and clinical products in the near future. Premarket regulatory scrutiny is, thus, an issue gaining considerable attention. Previous review papers have focused primarily on the toxicity of QDs. From the point of view of product regulation, however, parameters that determine exposure (e.g., dosage, transformation, transportation, and persistence) are just as important as inherent toxicity. We have structured our review paper according to regulatory risk assessment practices, in order to improve the utility of existing knowledge in a regulatory context. Herein, we summarize the state of academic knowledge on QDs pertaining not only to toxicity, but also their physicochemical properties, and their biological and environmental fate. We conclude this review with recommendations on how to tailor future research efforts to address the specific needs of regulators.

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

Bus, J. S., Becker, R. A. — Fri, 13 Nov 2009 13:24:06 PST

Modulation of Aflatoxin B1-Mediated Genotoxicity in Primary Cultures of Human Hepatocytes by Diindolylmethane, Curcumin, and Xanthohumols

Fri, 13 Nov 2009 13:24:06 PST

This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol.

Use of Short-term Transcriptional Profiles to Assess the Long-term Cancer-Related Safety of Environmental and Industrial Chemicals

Fri, 13 Nov 2009 13:24:06 PST

The process for evaluating chemical safety is inefficient, costly, and animal intensive. There is growing consensus that the current process of safety testing needs to be significantly altered to improve efficiency and reduce the number of untested chemicals. In this study, the use of short-term gene expression profiles was evaluated for predicting the increased incidence of mouse lung tumors. Animals were exposed to a total of 26 diverse chemicals with matched vehicle controls over a period of 3 years. Upon completion, significant batch-related effects were observed. Adjustment for batch effects significantly improved the ability to predict increased lung tumor incidence. For the best statistical model, the estimated predictive accuracy under honest fivefold cross-validation was 79.3% with a sensitivity and specificity of 71.4 and 86.3%, respectively. A learning curve analysis demonstrated that gains in model performance reached a plateau at 25 chemicals, indicating that the size of current data set was sufficient to provide a robust classifier. The classification results showed that a small subset of chemicals contributed disproportionately to the misclassification rate. For these chemicals, the misclassification was more closely associated with genotoxicity status than with efficacy in the original bioassay. Statistical models were also used to predict dose-response increases in tumor incidence for methylene chloride and naphthalene. The average posterior probabilities for the top models matched the results from the bioassay for methylene chloride. For naphthalene, the average posterior probabilities for the top models overpredicted the tumor response, but the variability in predictions was significantly higher. The study provides both a set of gene expression biomarkers for predicting chemically induced mouse lung tumors and a broad assessment of important experimental and analysis criteria for developing microarray-based predictors of safety-related end points.

S-Adenosylhomocysteine Promotes the Invasion of C6 Glioma Cells via Increased Secretion of Matrix Metalloproteinase-2 in Murine Microglial BV2 Cells

Lin, H.-C., Song, T.-Y., Hu, M.-L. — Fri, 13 Nov 2009 13:24:06 PST

S-Adenosylhomocysteine (SAH) is a risk factor for many diseases, including tumor progression and neurodegenerative disease. In this study, we examined the hypothesis that SAH may indirectly enhance the invasion of C6 glioma cells by induction of matrix metalloproteinase-2 (MMP-2) secreted from the murine microglia BV2 cells. We obtained conditioned medium (CM) by incubating BV2 cells with SAH (1–50nM) for 24 h. We found that the SAH-containing CM (SAH-BV2-CM) strongly enhanced the invasiveness of C6 glioma cells and that this effect increased with increasing concentrations of SAH in the SAH-BV2-CM. The effect of CM could be attributed to its MMP-2 activity, as a result of increased protein and messenger RNA expression of MMP-2 in BV2 cells induced by SAH. In BV2 cells treated with SAH, the binding abilities of nuclear factor-kappa B (NF-B) and stimulatory protein-1 (Sp1) to the MMP-2 promoter were increased, whereas the level of NF-B inhibitor was decreased. In addition, SAH significantly increased the phosphorylation of extracellular signal–regulated kinase (ERK) and phosphatidylinositol-3-kinase/serine/threonine protein kinase (or protein kinase B) (PI3K/Akt) proteins but did not affect that of c-Jun NH2-terminal kinase or p38. Pretreatment of BV2 cells with an inhibitor specific for ERK (U0126) markedly abated the expression of ERK and MMP-2. Furthermore, SAH significantly and dose dependently decreased tissue inhibitor of metalloproteinase-2 (TIMP-2) in BV2 cells. Thus, SAH may induce the invasiveness of C6 glioma cells by decreased TIMP-2 expression and increased MMP-2 expression in BV2 cells. The latter effect is likely mediated through the ERK and PI3K/Akt pathways, with increased binding activities of NF-B and Sp1 to the MMP-2 gene promoter.

Mal-Development of the Penis and Loss of Fertility in Male Rats Treated Neonatally with Female Contraceptive 17{alpha}-Ethinyl Estradiol: A Dose-Response Study and a Comparative Study with a Known Estrogenic Teratogen Diethylstilbestrol

Fri, 13 Nov 2009 13:24:06 PST

The objectives of this study were to find a minimal dose of 17-ethinyl estradiol (EE) that is detrimental to the developing penis and fertility and to compare estrogenic effects between EE and diethylstilbestrol (DES). Neonatal rats received EE at 10 ng (1 µg/kg), 100 ng, 1 µg, or 10 µg per pup on alternate days from postnatal days 1 to 11 (dose-response study) or received EE or DES at 100 ng per pup daily from postnatal days 1 to 6 (comparative study). Effects of EE were dose dependent, with ≥ 100-ng dose inducing significant (p < 0.05) reductions in penile length, weight, and diameter. Additionally, the penis was malformed, characterized by underdeveloped os penis and accumulation of fat cells. Fertility was 0% in the ≥ 1-µg groups, in contrast to 60% in the 100-ng group and 100% in the 10-ng and control groups. Animals treated with ≥ 10 ng had significant reductions in the weight of bulbospongious muscle, testis, seminal vesicle, epididymal fat pad, and in epididymal sperm numbers. A comparison of EE and DES effects showed similar reductions in penile weight and length and the weight of bulbospongiosus muscle, testis, seminal vesicle, epididymis, and epididymal fat pad in both adolescent and adult rats. While 5/6 control males sired, only 1/6 in the EE group and 0/6 in the DES group sired. Hence, neonatal exposure to EE at 10 ng (environmentally relevant dose) adversely affects male reproductive organs. A dose ten times higher than this leads to permanently mal-developed penis and infertility. Furthermore, EE and DES exposures show similar level of toxicity to male reproductive organs.

Dynamic Nature of Alterations in the Endocrine System of Fathead Minnows Exposed to the Fungicide Prochloraz

Fri, 13 Nov 2009 13:24:06 PST

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17β-estradiol (E2) in female fish. At a prochloraz water concentration of 30 µg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 µg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.

Gene and Protein Responses of Human Monocytes to Extracellular Cysteine Redox Potential

Go, Y.-M., Craige, S. E., Orr, M., Gernert, K. M., Jones, D. P. — Fri, 13 Nov 2009 13:24:06 PST

The redox potential of the major thiol/disulfide couple, cysteine (Cys) and its disulfide cystine (CySS), in plasma (E_hCys) is oxidized in association with oxidative stress, and oxidized E_hCys is associated with cardiovascular disease risk. In vitro exposure of monocytes to oxidized E_hCys increases expression of the proinflammatory cytokine, interleukin-1β (IL-1β), suggesting that E_hCys could be a mechanistic link between oxidative stress and chronic inflammation. Because cell membranes contain multiple Cys-rich proteins, which could be sensitive to E_hCys, we sought to determine whether E_hCys specifically affects proinflammatory signaling or has other effects on monocytes. We used microarray analysis and mass spectrometry–based proteomics to evaluate global changes in protein redox state, gene expression, and protein abundance in monocytes in response to E_hCys. Pathway analysis results revealed that in addition to IL-1β-related pathways, components of stress/detoxification and cell death pathways were increased by oxidized E_hCys, while components of cell growth and proliferation pathways were increased by a reduced potential. Phenotypic studies confirmed that a cell stress response occurred with oxidized E_h and that cell proliferation was stimulated with reduced E_h. Therefore, plasma E_hCys provides a control over monocyte phenotype, which could contribute to cardiovascular disease risk and provide a novel therapeutic target for disease prevention.

Establishment of an Immunoglobulin M Antibody-Forming Cell Response Model for Characterizing Immunotoxicity in Primary Human B Cells

Lu, H., Crawford, R. B., North, C. M., Kaplan, B. L. F., Kaminski, N. E. — Fri, 13 Nov 2009 13:24:06 PST

Rodent models have been extensively utilized to identify putative human immunotoxicants; however, even when immunotoxicity is established, uncertainty remains whether the effects are predictive of human risk. Therefore, the objective of this study was to establish a polyclonal immunoglobulin M (IgM) antibody-forming cell (AFC) response model to directly characterize immunotoxicity in primary mouse or human B cells. CD40 ligand (CD40L) was selected to activate B cells because it effectively drives both primary human and mouse B cells in vitro to AFC in a physiologically relevant manner to mimic T-cell-dependent antibody responses in vivo. In this model, the IgM AFC response is induced by cell surface–expressed CD40L and promoted by recombinant cytokines. Reported here are the conditions required to induce IgM AFC responses using mouse splenic B cells or human peripheral blood B cells, allowing for species comparisons. Moreover, less than one order of magnitude difference was observed in the CD40L-induced B-cell AFC responses based on data from multiple donors. In addition to antibody production, proliferation and phenotypic changes characteristic of B-cell activation as well as the plasma cell phenotype were also significantly induced. Finally, two well-characterized immunotoxicants, arsenic and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, using the CD40L-induced IgM AFC response were compared in both mouse and human B cells. Collectively, an IgM AFC response model is described that can be applied to assess the sensitivity of antibody responses to modulation by xenobiotics using mouse as well as human primary B cells.

Discrimination for Genotoxic and Nongenotoxic Carcinogens by Gene Expression Profiling in Primary Mouse Hepatocytes Improves with Exposure Time

Fri, 13 Nov 2009 13:24:06 PST

Assessing the potential carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability and are simultaneously expensive and long lasting. In order to seek for alternatives, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from nongenotoxic (NGTX) carcinogens class discrimination was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36, and 48 h with two GTX and two NGTX carcinogens. For validation, two additional GTX compounds were studied at 24 and 48 h. Immunostaining of H2AX foci was applied in order to phenotypically verify DNA damage. It confirmed significant induction of DNA damage after treatment with GTX compounds but not with NGTX compounds. Whole-genome gene expression modifications were analyzed by means of Affymetrix microarrays. When using differentially expressed genes from data sets normalized by Robust Multi-array Average, the two classes and various compounds were better separated from each other by hierarchical clustering when increasing the treatment period. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray improved with time and resulted in correct classification of the validation compounds. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating GTX from NGTX compounds and that this discrimination improves with increasing treatment period.

Weighted Feature Significance: A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features

Fri, 13 Nov 2009 13:24:06 PST

In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call weighted feature significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on the following: (1) data from quantitative high–throughput screening cytotoxicity and caspase activation assays conducted at the National Institutes of Health Chemical Genomics Center, (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program, and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances. Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency tested also at the National Institutes of Health Chemical Genomics Center. We compared the performance of our WFS approach with classical classification methods such as Naive Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.

Age-Dependent Susceptibility to Manganese-Induced Neurological Dysfunction

Fri, 13 Nov 2009 13:24:06 PST

Chronic exposure to manganese (Mn) produces a spectrum of cognitive and behavioral deficits associated with a neurodegenerative disorder resembling Parkinson’s disease. The effects of high-dose exposure to Mn in occupational cohorts and in adult rodent models of the disease are well described but much less is known about the behavioral and neurochemical effects of Mn in the developing brain. We therefore exposed C57Bl/6 mice to Mn by intragastric gavage as juveniles, adults, or both, postulating that mice exposed as juveniles and then again as adults would exhibit greater neurological and neurochemical dysfunction than mice not preexposed as juveniles. Age- and sex-dependent vulnerability to changes in locomotor function was detected, with juvenile male mice displaying the greatest sensitivity, characterized by a selective increase in novelty-seeking and hyperactive behaviors. Adult male mice preexposed as juveniles had a decrease in total movement and novelty-seeking behavior, and no behavioral changes were detected in female mice. Striatal dopamine levels were increased in juvenile mice but were decreased in adult preexposed as juveniles. Levels of Mn, Fe, and Cu were determined by inductively coupled plasma-mass spectrometry, with the greatest accumulation of Mn detected in juvenile mice in the striatum, substantia nigra (SN), and cortex. Only modest changes in Fe and Cu were detected in Mn-treated mice, primarily in the SN. These results reveal that developing mice are more sensitive to Mn than adult animals and that Mn exposure during development enhances behavioral and neurochemical dysfunction relative to adult animals without juvenile exposure.

Developmental Exposure to Manganese Increases Adult Susceptibility to Inflammatory Activation of Glia and Neuronal Protein Nitration

Moreno, J. A., Streifel, K. M., Sullivan, K. A., Legare, M. E., Tjalkens, R. B. — Fri, 13 Nov 2009 13:24:06 PST

Chronic exposure to manganese (Mn) produces a neurodegenerative disorder affecting the basal ganglia characterized by reactive gliosis and expression of neuroinflammatory genes including inducible nitric oxide synthase (NOS2). Induction of NOS2 in glial cells causes overproduction of nitric oxide (NO) and injury to neurons that is associated with parkinsonian-like motor deficits. Inflammatory activation of glia is believed to be an early event in Mn neurotoxicity, but specific responses of microglia and astrocytes to Mn during development remain poorly understood. In this study, we investigated the effect of juvenile exposure to Mn on the activation of glia and production of NO in C57Bl/6J mice, postulating that developmental Mn exposure would lead to heightened sensitivity to gliosis and increased expression of NOS2 in adult mice exposed again later in life. Immunohistochemical analysis indicated that Mn exposure caused increased activation of both microglia and astrocytes in the striatum (St), globus pallidus (Gp), and substantia nigra pars reticulata (SNpr) of treated mice compared with controls. More robust activation of microglia was observed in juveniles, whereas astrogliosis was more prominent in adult mice preexposed during development. Co-immunofluorescence studies demonstrated increased expression of NOS2 in glia located in the Gp and SNpr. Additionally, greater increases in the level of 3-nitrotyrosine protein adducts were detected in dopamine- and cAMP-regulated phosphoprotein-32–positive neurons of the St of Mn-treated adult mice preexposed as juveniles. These data indicate that subchronic exposure to Mn during development leads to temporally distinct patterns of glial activation that result in elevated nitrosative stress in distinct populations of basal ganglia neurons.

Probucol Increases Glutathione Peroxidase-1 Activity and Displays Long-Lasting Protection against Methylmercury Toxicity in Cerebellar Granule Cells

Fri, 13 Nov 2009 13:24:06 PST

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here, we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase, isozyme 1 (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen peroxide or tert-butyl peroxide and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake, and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated with increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants, ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (1) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (2) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.

Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

Drechsel, D. A., Patel, M. — Fri, 13 Nov 2009 13:24:06 PST

Exposure to environmental pesticides can cause significant brain damage and has been linked with an increased risk of developing neurodegenerative disorders, including Parkinson's disease. Bipyridyl herbicides, such as paraquat (PQ), diquat (DQ), and benzyl viologen (BV), are redox cycling agents known to exert cellular damage through the production of reactive oxygen species (ROS). We examined the involvement of the mitochondrial respiratory chain in ROS production by bipyridyl herbicides. In isolated rat brain mitochondria, H₂O₂ production occurred with the following order of potency: BV > DQ > PQ in accordance with their measured ability to redox cycle. H₂O₂ production was significantly attenuated in all cases by antimycin A, an inhibitor of complex III. Interestingly, at micromolar (≤ 300µM) concentrations, PQ-induced H₂O₂ production was unaffected by complex I inhibition via rotenone, whereas DQ-induced H₂O₂ production was equally attenuated by inhibition of complex I or III. Moreover, complex I inhibition decreased BV-induced H₂O₂ production to a greater extent than with PQ or DQ. These data suggest that multiple sites within the respiratory chain contribute to H₂O₂ production by redox cycling bipyridyl herbicides. In primary midbrain cultures, H₂O₂ differed slightly with the following order of potency: DQ > BV > PQ. In this model, inhibition of complex III resulted in roughly equivalent inhibition of H₂O₂ production with all three compounds. These data identify a novel role for complex III dependence of mitochondrial ROS production by redox cycling herbicides, while emphasizing the importance of identifying mitochondrial mechanisms by which environmental agents generate oxidative stress contributing to parkinsonism.

Metallothionein-III Provides Neuronal Protection through Activation of Nuclear Factor-{kappa}B via the TrkA/Phosphatidylinositol-3 kinase/Akt Signaling Pathway

Fri, 13 Nov 2009 13:24:06 PST

Metallothionein (MT)-III is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. The present study investigated the mechanisms of MT-III protection of neuronal cells from hypoxia or DNA damage–induced cell death. MT-III reduced the hydrogen peroxide– or DNA damage–induced effects on neuronal cells, including the cell death, the activation of caspase-3 and -9, and the release of mitochondrial cytochrome c to the cytoplasm in a dose-dependent manner. MT-III also increased the activation of Akt, the phosphorylation and degradation of IB, the nuclear translocation/accumulation and the transcriptional activity of nuclear factor-B (NF-B) in neuronal cells in a dose-dependent manner. The MT-III–induced antiapoptotic effects and increase in NF-B activity were blocked by specific inhibitors of TrkA, phosphatidylinositol-3 kinase (PI3K), Akt, or NF-B, indicating that MT-III provides neuronal protection by activating NF-B through the TrkA/PI3K/Akt signaling pathway.

Early-Life Exposure to Dimethoate-Induced Reproductive Toxicity: Evaluation of Effects on Pituitary-Testicular Axis of Mice

Verma, R., Mohanty, B. — Fri, 13 Nov 2009 13:24:06 PST

In utero and lactational exposure to organophosphate dimethoate exerted toxic impact on the reproductive system of male mice. Pregnant mice were exposed to 4, 8, and 16 mg/kg of the pesticide, the sublethal doses (2.5, 5, and 10% of Lethal Dose₅₀ [LD₅₀]), via gavaging from gestation day (GD) 6 to postnatal day (PND) 21. The effects on the male reproductive system were evaluated at two stages: at prepubertal age (PND 22) and at the postpubertal age of PND 63. Gonadal inhibition was reflected in the significant reduction of weight and distinct histopathological alteration of testis and epididymis as well as in decreased sperm counts, which could be linked to hormonal imbalance caused by dimethoate interference of reproductive axis. Disruption of pituitary-testicular axis was shown in the weak immunointensity of luteinizing hormone (LH) cells, reduction in their size and number, and lowered plasma LH and testosterone levels as observed in the neonates exposed to two higher tested doses. In addition, the direct toxic impact of the pesticide on the testicular Leydig cells and inhibition of steroidogenesis could be suggested. Drastic reduction in the testosterone level (~70%) was suggestive of this effect. The adverse effects were persisted in the young adult mice. Developmental toxicity was evident in the highest dose-exposed (10% LD₅₀) group where GD length and stillbirths were significantly increased along with a decrease of body weight and anogenital distance of the fetus. Maternal exposure of pesticide during gestation and lactation periods thus adversely affected the pituitary-testicular axis of mice neonates, which further caused reproductive dysfunctioning of young adult mice.

Reproductive and Sphingolipid Metabolic Effects of Fumonisin B1 and its Alkaline Hydrolysis Product in LM/Bc Mice: Hydrolyzed Fumonisin B1 Did Not Cause Neural Tube Defects

Voss, K. A., Riley, R. T., Snook, M. E., Waes, J. G.-v. — Fri, 13 Nov 2009 13:24:06 PST

Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins are converted to their hydrolyzed analogs by alkaline cooking (nixtamalization). Both fumonisins and hydrolyzed fumonisins are found in nixtamalized foods such as tortillas, and consumption of tortillas has been implicated as a risk factor for neural tube defects (NTD). Fumonisin B₁ (FB₁) induced NTD when given (ip) to pregnant LM/Bc mice; however, neither the NTD induction potential of hydrolyzed fumonisin B₁ (HFB₁) nor its affect on sphingolipid metabolism in pregnant mice have been reported. The teratogenic potential of FB₁ and HFB₁ was therefore compared using the LM/Bc mouse model. Dams were dosed (ip) with 2.5, 5.0, 10, or 20 mg/kg (≤ 49 µmol/kg) body weight (bw) HFB₁ on embryonic day (E)7–E8. Negative and positive control groups were given vehicle or 10 mg/kg (14 µmol/kg) bw FB₁, respectively. The high dose of HFB₁ disrupted sphingolipid metabolism, albeit slightly, but did not cause maternal liver lesions or NTD (n = 8–10 litters per group). In contrast, 10 mg/kg bw FB₁ markedly disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the dams, increased fetal death rates, and decreased fetal weights. Furthermore, NTD were found in all FB₁-exposed litters (n = 10), and 66 ± 24% of the fetuses were affected. The findings indicate that HFB₁ does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupts sphingolipid metabolism at doses up to sevenfold higher (micromole per kilogram body weight basis) than the previously reported lowest observed adverse effect level for FB₁.

Inhalation Toxicity of Multiwall Carbon Nanotubes in Rats Exposed for 3 Months

Fri, 13 Nov 2009 13:24:06 PST

Carbon nanotubes (CNT) are of great commercial interest. Theoretically, during processing and handling of CNT and in abrasion processes on composites containing CNT, inhalable CNT particles might be set free. For hazard assessment, we performed a 90-day inhalation toxicity study with a multiwall CNT (MWCNT) material (Nanocyl NC 7000) according to Organisation for Economic Co-operation and Development test guideline 413. Wistar rats were head-nose exposed for 6 h/day, 5 days/week, 13 weeks, total 65 exposures, to MWCNT concentrations of 0 (control), 0.1, 0.5, or 2.5 mg/m³. Highly respirable dust aerosols were produced with a proprietary brush generator which neither damaged the tube structure nor increased reactive oxygen species on the surface. Inhalation exposure to MWCNT produced no systemic toxicity. However, increased lung weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5 mg/m³. These effects were accompanied by slight blood neutrophilia at 2.5 mg/m³. Incidence and severity of the effects were concentration related. At 0.1 mg/m³, there was still minimal granulomatous inflammation in the lung and in lung-associated lymph nodes; a no observed effect concentration was therefore not established in this study. The test substance has low dust-forming potential, as demonstrated by dustiness measurements, but nonetheless strict industrial hygiene measures must be taken during handling and processing. Toxicity and dustiness data such as these can be used to compare different MWCNT materials and to select the material with the lowest risk potential for a given application.

Acute Morphological and Toxicological Effects in a Human Bronchial Coculture Model after Sulfur Mustard Exposure

Fri, 13 Nov 2009 13:24:06 PST

Sulfur mustard (SM) is a strong alkylating agent. Inhalation of SM causes acute lung injury accompanied by severe disruption of the airway barrier. In our study, we tested the acute effects after mustard exposure in an in vitro coculture bronchial model of the proximal barrier. To achieve this, we seeded normal human bronchial epithelial explant-outgrowth cells (HBEC) together with lung fibroblasts as a bilayer on filter plates and exposed the bronchial model after 31 days of differentiation to various concentrations of SM (30, 100, 300, and 500µM). The HBEC formed confluent layers, expressing functional tight junctions as measured by transepithelial electrical resistance (TER). Mucus production and cilia formation reappeared in the coculture model. TER was measured after 2 and 24 h following treatment. Depending on the different concentrations, TER decreased in the first 2 h up to 55% of the control at the highest concentration. After 24 h, TER seemed to recover because at concentrations up to 300µM values were equal to the control. SM induced a widening of intercellular spaces and a loss in cell-matrix adhesion. Mucus production increased with the result that cilia ceased to beat. Changes in the proinflammatory cytokines interleukin (IL)-6 and IL-8 were also observed. Apoptotic markers such as cytochrome c, p53, Fas-associated protein with death domain, and procaspase-3 were significantly induced at concentrations of less than 100µM. In summary, SM induces morphological and biochemical changes that reflect pathological effects of SM injury in vivo. It is hoped to use this coculture model to understand further the pathogenesis of SM-induced barrier injury and to search for novel approaches in SM therapy.

Estimates of Cancer Potency of 2,3,7,8-Tetrachlorodibenzo(p)dioxin Using Linear and Nonlinear Dose-Response Modeling and Toxicokinetics

Simon, T., Aylward, L. L., Kirman, C. R., Rowlands, J. C., Budinsky, R. A. — Fri, 13 Nov 2009 13:24:06 PST

Linear and nonlinear toxicity criteria were derived for 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) using the recent National Toxicology Program rat cancer bioassay. Dose-response relationships were assessed for combined liver tumors based on lifetime average liver concentrations (LALCs) estimated with a toxicokinetic model. Rat LALC estimates at the 1% point of departure (POD) were obtained with benchmark dose (BMD) modeling to yield the BMD₀₁ in terms of LALC. The same toxicokinetic model was used to back-extrapolate the human-equivalent external dose (HED). A linear cancer slope factor (CSF) with a value of 1 x 10^–4 per pg/kg/day was calculated as the ratio between the benchmark response rate and the HED at the lower confidence limit of the benchmark dose (BMDL)₀₁. A nonlinear reference dose (RfD) with a value of 100 pg/kg/day was developed from the BMD₀₁ value by applying uncertainty factors to rat internal and human external doses. The RfD was 100 times higher than the 10^–4 risk-specific dose (RSD) based on the linear CSF. For comparison, BMD₀₁ and BMDL₀₁ values were developed for key events in the tumor promotion mode of action (MOA) of TCDD. This MOA involves dysregulation of the normal function of the aryl hydrocarbon receptor and its associated biological processes and results in pathologies that drive tumor promotion and progression. The BMD₀₁ values for key events were consistent with the timing of the key events within the MOA and provided support for the choices of the 1% tumor rate as a POD and dichotomous Hill model for representing receptor-mediated carcinogenicity. Because a threshold toxicity criterion most accurately reflects the MOA, the RfD for TCDD with a value of 100 pg/kg/day is considered appropriate for regulatory purposes, consistent with a 2006 NRC panel's recommendation to develop a threshold-based cancer potency factor for TCDD and with the methodology in U.S. Environmental Protection Agency's Cancer Guidelines.

Toxicogenomics Applied to Cultures of Human Hepatocytes Enabled an Identification of Novel Petasites hybridus Extracts for the Treatment of Migraine with Improved Hepatobiliary Safety

Anderson, N., Meier, T., Borlak, J. — Fri, 13 Nov 2009 13:24:06 PST

Butterbur extracts (Petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive of rare hepatobiliary toxicity. To evaluate its hepatotoxic potential, a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic C_max levels of 60 ng/ml. Nonetheless, in a 28-day toxicity study at ~200-fold of therapeutic doses, induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-month chronic toxicity study, the initial hepatobiliary effects were reproduced, but at the end of the study, liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at > 170-fold of therapeutic C_max levels, including cytotoxicity (lactate dehydrogenase, MTT, and ATP), transaminase activities (alanine aminotransferase and aspartate aminotransferase), albumin synthesis, urea and testosterone metabolism to assay for cytochrome P450 monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high and well above therapeutic doses, liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport and lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore reemphasizing the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.

High-Mobility Group Box-1 Protein and Keratin-18, Circulating Serum Proteins Informative of Acetaminophen-Induced Necrosis and Apoptosis In Vivo

Fri, 13 Nov 2009 13:24:06 PST

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

A Single Exposure to Particulate or Gaseous Air Pollution Increases the Risk of Aconitine-Induced Cardiac Arrhythmia in Hypertensive Rats

Hazari, M. S., Haykal-Coates, N., Winsett, D. W., Costa, D. L., Farraj, A. K. — Fri, 13 Nov 2009 13:24:06 PST

Epidemiological studies demonstrate an association between arrhythmias and air pollution. Aconitine-induced cardiac arrhythmia is widely used experimentally to examine factors that alter the risk of arrhythmogenesis. In this study, Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats acutely exposed to synthetic residual oil fly ash (s-ROFA) particles (450 µg/m³) were "challenged" with aconitine to examine whether a single exposure could predispose to arrhythmogenesis. Separately, SH rats were exposed to varied particulate matter (PM) concentrations (0.45, 1.0, or 3.5 mg/m³ s-ROFA), or the irritant gas acrolein (3 ppm), to better assess the generalization of this challenge response. Rather than directly cause arrhythmias, we hypothesized that inhaled air pollutants sensitize the heart to subsequent dysrhythmic stimuli. Twenty-four hour postexposure, urethane-anesthetized rats were monitored for heart rate (HR), electrocardiogram, and blood pressure (BP). SH rats had higher baseline HR and BP and significantly longer PR intervals, QRS duration, QTc, and JTc than WKY rats. PM exposure caused a significant increase in the PR interval, QRS duration, and QTc in WKY rats but not in SH rats. Heart rate variability was significantly decreased in WKY rats after PM exposure but increased in SH rats. Cumulative dose of aconitine that triggered arrhythmias in air-exposed SH rats was lower than WKY rats and even lower for each strain postexposure. SH rats exposed to varied concentrations of PM or acrolein developed arrhythmia at significantly lower doses of aconitine than controls; however, there was no PM concentration–dependent response. In conclusion, a single exposure to air pollution may increase the sensitivity of cardiac electrical conduction to disruption. Moreover, there seem to be host factors (e.g., cardiovascular disease) that increase vulnerability to triggered arrhythmias regardless of the pollutant or its concentration.

Tue, 27 Oct 2009 12:53:42 PDT

COVER

Tue, 27 Oct 2009 12:53:42 PDT

Editorial Board

Tue, 27 Oct 2009 12:53:42 PDT

SUBSCRIPTIONS

Tue, 27 Oct 2009 12:53:42 PDT

Hypoxia Response: A Model Toxicity Pathway for High-Throughput Screening

Simmons, S. O. — Tue, 27 Oct 2009 12:53:42 PDT

Nitrative and Oxidative Stress in Toxicology and Disease

Tue, 27 Oct 2009 12:53:43 PDT

Persistent inflammation and the generation of reactive oxygen and nitrogen species play pivotal roles in tissue injury during disease pathogenesis and as a reaction to toxicant exposures. The associated oxidative and nitrative stress promote diverse pathologic reactions including neurodegenerative disorders, atherosclerosis, chronic inflammation, cancer, and premature labor and stillbirth. These effects occur via sustained inflammation, cellular proliferation and cytotoxicity and via induction of a proangiogenic environment. For example, exposure to the ubiquitous air pollutant ozone leads to generation of reactive oxygen and nitrogen species in lung macrophages that play a key role in subsequent tissue damage. Similarly, studies indicate that genes involved in regulating oxidative stress are altered by anesthetic treatment resulting in brain injury, most notable during development. In addition to a role in tissue injury in the brain, inflammation, and oxidative stress are implicated in Parkinson's disease, a neurodegenerative disease characterized by the loss of dopamine neurons. Recent data suggest a mechanistic link between oxidative stress and elevated levels of 3,4-dihydroxyphenylacetaldehyde, a neurotoxin endogenous to dopamine neurons. These findings have significant implications for development of therapeutics and identification of novel biomarkers for Parkinson's disease pathogenesis. Oxidative and nitrative stress is also thought to play a role in creating the proinflammatory microenvironment associated with the aggressive phenotype of inflammatory breast cancer. An understanding of fundamental concepts of oxidative and nitrative stress can underpin a rational plan of treatment for diseases and toxicities associated with excessive production of reactive oxygen and nitrogen species.

Endless Possibilities: Stem Cells and the Vision for Toxicology Testing in the 21st Century

Chapin, R. E., Stedman, D. B. — Tue, 27 Oct 2009 12:53:43 PDT

The National Research Council's (NRC) toxicity testing vision lays out a bold future for our field. It depends heavily on computational algorithms based on the latest knowledge of cellular biochemistry and protein interaction pathways, exposing human cells to novel compounds in vitro, and being able to understand the changes seen. At the same time, significant strides are being made in our understanding of the control, production, and "behavior" of stem cells. While stem cells offer seemingly limitless possibilities for regenerative medicine, they have already delivered new assays to predict embryo-fetal developmental toxicity in vitro. In addition to providing a model of cells undergoing differentiation and proliferation, stem cells will play a major role by giving rise to many of the differentiated cell types on which this new vision depends. These will not be pure populations of single cell types but mixtures of cells much more representative of tissues in vitro. Moving from cells alone in a culture dish toward the more physiological condition of multiple cell types being able to interact to maintain homeostasis in the face of a disequilibrating force (like a toxic exposure) will lead us toward more useful and correct predictions of in vivo toxicities. Despite the seemingly insurmountable hurdles, persistence and creativity are on our side. We expect that a long series of successive iterations of predictive models will eventually yield a working process that approximates the NRC's vision and delivers on the promise of faster evaluation of chemicals with reduced animal use.

Lactational Transfer of Manganese in Rats: Predicting Manganese Tissue Concentration in the Dam and Pups from Inhalation Exposure with a Pharmacokinetic Model

Tue, 27 Oct 2009 12:53:43 PDT

Manganese (Mn) is an essential element. However, excess Mn causes neurotoxicity. Fetuses and neonates have been discussed as potentially sensitive subpopulations for Mn. In the present study, a previously published physiologically based pharmacokinetic model for Mn in adult rats was extended to examine exposure conditions that could lead to increased central nervous system Mn in developing rats. The basic structure had saturable tissue binding, homeostatic control of uptake and excretion, and tissue-specific increases in Mn from inhalation. Modifications made for lactating dam and pups included differential tissue-binding capacities in developing pups, increased absorption of dietary Mn in lactating dam, and more efficient gastrointestinal absorption and lower basal biliary excretion in pups. Enhancement of biliary excretion in pups was also required to accurately simulate tissue Mn during early postnatal inhalation. Overall, these changes were concordant with the biology of Mn and other essential metals during development. The resulting model simulations match a variety of published studies on maternal Mn homeostasis during lactation, milk Mn levels, and changing patterns of neonatal tissue Mn for normal dietary intake and with Mn inhalation. Our successful description of Mn kinetics across these life stages suggests that the present model can help describe the relationship between dose of exposure and target tissue Mn concentrations across different developmental stages and its potential risks and assess whether infants and children should be regarded as susceptible populations for Mn inhalation.

Evaluating Placental Transfer and Tissue Concentrations of Manganese in the Pregnant Rat and Fetuses after Inhalation Exposures with a PBPK Model

Tue, 27 Oct 2009 12:53:43 PDT

A Physiologically Based Pharmaco Kinetic (PBPK) model, based on a published description of manganese (Mn) kinetics in adult rats, has been developed to describe Mn uptake and tissue distribution in the pregnant dam and fetus during dietary and inhalation exposures. This extension incorporated key physiological processes controlling Mn pharmacokinetics during pregnancy and fetal development. After calibration against tissue Mn concentrations observed during late gestation, the model accurately simulated Mn tissue distribution in the dam and fetus following both diet and inhalation exposures to the pregnant rat. Maternal to fetal transfer of Mn through placenta was described using two pathways: a saturable active transport with high affinity and a simple diffusion. The active transport dominates at basal and lower Mn exposure, whereas at higher Mn exposure, the relative contribution of the diffusion pathway increases. To simulate fetal tissue Mn, tissue-binding parameters and preferential influx/efflux rates in fetal brain were adjusted from the adult model based on differential developmental processes and varying tissue demands for Mn in early life. Model simulations were consistent with observed tissue Mn concentrations in fetal tissues, including brain for diet alone and for combined diet and inhalation. Simulations of Mn in placenta and other maternal tissues in late gestation correlated well with measured tissue concentrations. This model, together with our published models for Mn kinetics during lactation and postnatal development, will help to address concerns about Mn neurotoxicity in potentially sensitive human subpopulation, such as infants and children by providing an estimate of Mn exposure in the population of interest.

3-Methylindole is Mutagenic and a Possible Pulmonary Carcinogen

Tue, 27 Oct 2009 12:53:43 PDT

Previous work has shown that bioactivation of the cigarette smoke pneumotoxicant 3-methylindole (3MI) by pulmonary cytochrome P450 enzymes is directly associated with formation of DNA adducts. Here, we present evidence that normal human lung epithelial cells, exposed to low micromolar concentrations of 3MI, showed extensive DNA damage, as measured by the comet assay, with similar potency to the prototypical genotoxic agents, doxorubicin and irinotecan. The DNA damage caused by 3MI was predominantly caused by single-strand breaks. Furthermore, we show that this damage decreased with time, given a subtoxic concentration, with detectable DNA fragmentation peaking 4 h after exposure and diminishing to untreated levels within 24 h. Pretreatment with an inhibitor of poly(ADP-ribose) polymerase 1 (PARP1), NU1025, nearly doubled the DNA damage produced by 5µM 3MI, implying that PARP1, which among other activities, functions to repair single-strand breaks in DNA, repaired at least some of the 3MI-induced DNA fragmentation. A key cellular response to DNA damage, phosphorylation, and nuclear localization of p53 was seen at subtoxic levels of 3MI exposure. 3MI was highly mutagenic, with essentially the same potency as the prototype carcinogen, benzo[a]pyrene, only when a lung-expressed CYP2F3 enzyme was used to dehydrogenate 3MI to its putative DNA-alkylating intermediate. Conversely, a rat liver S9 metabolic system did not bioactivate 3MI to its mutagenic intermediate(s). Concentrations higher than 25µM caused apoptosis, which became extensive at 100µM, similar to the response seen with 10µM doxorubicin. Our findings indicate that there is a low concentration window in which 3MI can cause extensive DNA damage and mutation, without triggering apoptotic defenses, reinforcing the hypothesis that inhaled 3MI from cigarette smoke may be a potent lung-selective carcinogen.

An Evaluation of Estrogenic Activity of Parabens Using Uterine Calbindin-D9k Gene in an Immature Rat Model

Vo, T. T. B., Jeung, E.-B. — Tue, 27 Oct 2009 12:53:43 PDT

In the present study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutylparaben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17-ethinylestradiol (EE, 1 mg/kg body weight [BW]/day) or parabens (62.5, 250, and 1000 mg/kg BW/day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the highest dose of isopropyl-, butyl-, and isobutylparaben. In addition, these parabens induced uterine CaBP-9k messenger RNA (mRNA) and protein levels, whereas cotreatment of parabens and fulvestrant, a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ER- and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER- mRNA and protein levels, whereas cotreatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER- expression, suggesting that CaBP-9k may not express via ER- pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER-regulating gene, at both transcriptional and translational levels was indicated in the highest dose of isopropyl- and butylparaben. These parabens-induced PR gene expression was completely blocked by fulvestrant. This result indicates that CaBP-9k expression may involve with PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which may be mediated by a PR and/or ER- signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

Chlorotriazine Herbicides and Metabolites Activate an ACTH-dependent Release of Corticosterone in Male Wistar Rats

Tue, 27 Oct 2009 12:53:43 PDT

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

Characterization of the Hypothalamic-Pituitary-Adrenal Axis Response to Atrazine and Metabolites in the Female Rat

Tue, 27 Oct 2009 12:53:44 PDT

Atrazine (ATR) has recently been shown to activate the hypothalamic-pituitary-adrenal (HPA) axis in rodents. The current study investigated the effect of ATR and two of its chlorinated metabolites, desisopropylatrazine (DIA) and diamino-s-chlorotriazine (DACT), on the HPA axis in the Long-Evans female rat. A single oral gavage administration of 75 mg/kg ATR or 60.2 mg/kg DIA (a dose equimolar to the applied ATR dose) during the morning of proestrus resulted in significant, acute increases in circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Oral doses of ATR or DIA were given daily over the course of the 4-day ovarian cycle starting on the day of vaginal estrus, resulted in a similar, dose-responsive activation of the HPA axis. The increase in ACTH, corticosterone, and progesterone by these compounds was of a similar magnitude to that produced by 5-min restraint stress. Single or multiple oral exposures to DACT, on the other hand, did not significantly alter pituitary-adrenal hormone release. These results were observed despite plasma levels of DACT being higher than any other metabolite at the time of hormone measurement. Overall, circulating metabolite concentrations following equimolar dosing were much higher than those observed after ATR administration. Additional studies indicated that the activation of the HPA axis by oral exposure to ATR and DIA was not due simply to the stimulation of gastrointestinal afferents. Similar responses were observed in rats which received an oral dose of ATR following bilateral subdiaphramatic vagotomy and following intravenous administration of DIA in jugular vein catheterized animals. We conclude that ATR and the metabolite DIA significantly activate the HPA axis following oral exposure in the female rat. Activation of this endocrine axis by these chlorotriazines could contribute to the induced changes of female reproductive function reported previously.

Comparative Study on Susceptibility to 1-Bromopropane in Three Mice Strains

Liu, F., Ichihara, S., Mohideen, S. S., Sai, U., Kitoh, J., Ichihara, G. — Tue, 27 Oct 2009 12:53:44 PDT

Previous studies indicate that 1-bromopropane (1BP) has neurotoxicity and reproductive toxicity both in humans and animals. The present study investigated strain differences in susceptibility to 1BP and identified possible biological factors that determine such susceptibility. Twenty-four male mice of each of the three strains (C57BL/6J, DBA/2J, and BALB/cA) were divided into four groups of six each and exposed to 1BP at 0, 50, 110, and 250 ppm for 8 h/day for 28 days by inhalation. At the end of exposure period, the relative susceptibilities of each strain to 1BP-mediated hepatotoxicity and male reproductive toxicity were evaluated. The contributing factors to strain-dependent susceptibility were assessed by determination of hepatic CYP2E1 levels, glutathione-S-transferase (GST) activity, glutathione (GSH) status, and NAD(P)H:quinone oxidoreductase and heme oxygenase-1 mRNA levels. Liver histopathology showed significantly larger area of liver necrosis and more degenerative lobules in BALB/cA in the order of BALB/cA > C57BL/6J > DBA/2J. BALB/cA showed higher CYP2E1 protein level and lower total GSH content and GST activity in the liver than DBA/2J. These results indicate that BALB/cA mice are the most susceptible to hepatotoxicity of 1BP among the three strains tested, and that CYP2E1, GSH level/GST activity may contribute to the susceptibility to 1BP hepatotoxicity. Exposure to ≥ 50 ppm of 1BP also decreased sperm count and sperm motility and increased sperms with abnormal heads in all three strains mice in a dose-dependent manner. Comparison with previous studies in rats indicates that mice are far more susceptible than rats to 1BP regarding hepatotoxicity and reproductive toxicity.

Urban Particulate Matter Causes ER Stress and the Unfolded Protein Response in Human Lung Cells

Watterson, T. L., Hamilton, B., Martin, R., Coulombe, R. A. — Tue, 27 Oct 2009 12:53:44 PDT

Because of its presumed adverse health effects, particulate air pollution (PM) has received growing attention, but the cellular mechanisms by which PM exerts toxicity are not well elucidated. PM has been associated with early mortality from illnesses that share endoplasmic reticulum (ER) stress as a mechanism of pathogenesis. In this study, we examined whether PM would induce the unfolded protein response (UPR) which is a cellular response to ER stress. Coarse (PM₁₀) and fine (PM_2.5) PM was collected from a single location in Northern Utah's Cache Valley during atmospheric inversions occurring in January 2002 and January 2003. Extracts of PM samples were added (12.5 and 25 µg/ml) to cultured human bronchial epithelial (BEAS-2B) cells for 24 h. At these concentrations neither PM nor LPS exhibited demonstrable cytotoxicity by the neutral red assay. However, PM elicited significant increases of unfolded protein response (UPR)–related post-translational modifications, such as S6 ribosomal protein, heat-shock protein (Hsp)27, and protein kinase related protein phosphorylation and cleavage of activating transcription factor (ATF)-6. PM exposure also resulted in significant increases in the UPR-associated proteins ATF-4, Hsp70, Hsp90, and binding immunoglobulin protein. PM also interfered with the export of Hsp70 from the cells in a concentration-dependent manner and resulted in release of C-reactive protein. Calpain was upregulated and activated in PM-treated cultures, though these events were not proapoptotic. This study demonstrates that PM is capable of inducing ER stress and the UPR in vitro and may be a mechanism by which PM exerts toxicity.

Simultaneous In Vivo Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS-stimulated Primary IgM Response

North, C. M., Crawford, R. B., Lu, H., Kaminski, N. E. — Tue, 27 Oct 2009 12:53:44 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent suppressor of humoral immunity but the specific molecular mechanisms responsible for immunosuppression by TCDD are poorly understood. In vivo and in vitro studies of the primary humoral IgM response demonstrated that the B cell is a sensitive cell type to modulation by TCDD. We hypothesized that in vivo administration of TCDD disrupts expression of transcription factors controlling B cell to plasma cell differentiation. Female C57BL6 mice were treated with a single dose of TCDD (3, 10, or 30 µg/kg) and/or vehicle (sesame oil). On day 4 post-TCDD administration mice were sensitized with 25 µg lipopolysacchride (LPS) by intraperitioneal injection to stimulate an immune response. Splenocytes were isolated on subsequent days following LPS, up to 3 days post-LPS, and the expression of IgM, XBP-1, PAX5, BCL-6, and Blimp-1 was assessed. TCDD treatment dose-dependently suppressed LPS-induced IgM antibody-forming cell number, which was correlated with decreased frequency of CD19⁺ CD138⁺ cells. Gene expression analysis revealed that TCDD caused a dose-dependent suppression of Igµ chain, Ig chain, IgJ chain, XBP-1, and Blimp-1. TCDD also dose-dependently suppressed LPS-stimulated increases in Blimp-1 protein expression in CD19⁺ B cells. The deregulation of Blimp-1 expression by TCDD provides a partial explanation for the concomitant suppression of the IgM response and confirms previous observations established in vitro.

Gestational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Disrupts B-Cell Lymphopoiesis and Exacerbates Autoimmune Disease in 24-Week-Old SNF1 Mice

Tue, 27 Oct 2009 12:53:44 PDT

Female SNF₁ hybrid mice spontaneously develop an immune complex–mediated glomerulonephritis as early as 24 weeks of age, whereas the disease onset in males is much slower. Further, a rise in concentration of glomerulus-specific autoantibodies via autoreactive B cells is critical to progression of the disease in this strain. Environmental factors contributing to the onset or degree of such autoimmunity are of interest yet poorly understood. In the present study, time-pregnant SWR x NZB dams (10/treatment) were gavaged on gestational 12 with 40 or 80 mg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the SNF₁ offspring were evaluated at 24 weeks of age. Bone marrow B220^lowCD24^–AA4.1⁺ committed B lineage progenitors were increased in female offspring by TCDD, however, committed progenitors and pro-B cells were decreased in males. Splenic marginal zone B cells (CD21^hiCD24^low–int) were decreased and follicular B cells (CD21^intCD24^low) were increased across sex by prenatal TCDD, whereas transitional-2 B cells (CD21^intCD24^hi) and (CD23^low–int CD1^low–int) were decreased in males only. Antibodies to double-stranded DNA were significantly increased across sex by TCDD. Anti-IgG and anti-C3 immune complex renal deposition was visibly worsened in females, and present in TCDD-treated males. These data suggest that developmental exposure to TCDD permanently and differentially alters humoral immune function by sex, and exacerbates a type III hypersensitivity lupus-like autoimmune disease in genetically predisposed mice.

Mast Cells Mediate the Immune Suppression Induced by Dermal Exposure to JP-8 Jet Fuel

Limon-Flores, A. Y., Chacon-Salinas, R., Ramos, G., Ullrich, S. E. — Tue, 27 Oct 2009 12:53:44 PDT

Applying jet propulsion-8 (JP-8) jet fuel to the skin of mice induces immune suppression. Applying JP-8 to the skin of mice suppresses T-cell–mediated immune reactions including, contact hypersensitivity (CHS) delayed-type hypersensitivity and T-cell proliferation. Because dermal mast cells play an important immune regulatory role in vivo, we tested the hypothesis that mast cells mediate jet fuel–induced immune suppression. When we applied JP-8 to the skin of mast cell deficient mice CHS was not suppressed. Reconstituting mast cell deficient mice with wild-type bone marrow derived mast cells (mast cell "knock-in mice") restored JP-8–induced immune suppression. When, however, mast cells from prostaglandin E₂ (PGE₂)–deficient mice were used, the ability of JP-8 to suppress CHS was not restored, indicating that mast cell–derived PGE₂ was activating immune suppression. Examining the density of mast cells in the skin and lymph nodes of JP-8-treated mice indicated that jet fuel treatment caused an initial increase in mast cell density in the skin, followed by increased numbers of mast cells in the subcutaneous space and then in draining lymph nodes. Applying JP-8 to the skin increased mast cell expression of CXCR4, and increased the expression of CXCL12 by draining lymph node cells. Because CXCL12 is a chemoattractant for CXCR4+ mast cells, we treated JP-8-treated mice with AMD3100, a CXCR4 antagonist. AMD3100 blocked the mobilization of mast cells to the draining lymph node and inhibited JP-8–induced immune suppression. Our findings demonstrate the importance of mast cells in mediating jet fuel–induced immune suppression.

Identification of Chemical Compounds that Induce HIF-1{alpha} Activity

Tue, 27 Oct 2009 12:53:45 PDT

Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1 and constitutively expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1 inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1 activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

Investigation of Peptide Reactivity of Pro-hapten Skin Sensitizers Using a Peroxidase-Peroxide Oxidation System

Tue, 27 Oct 2009 12:53:45 PDT

Skin protein reactivity is a well established key step in the development of skin sensitization. Understanding the relationship between a chemical's ability to react with or modify skin protein and skin sensitization has led to the development of the Direct Peptide Reactivity Assay (DPRA) in our laboratory. A current limitation of the DPRA is that it cannot readily measure the reactivity of pro-hapten chemical sensitizers. Pro-haptens are chemical sensitizers that are not directly reactive and must be bioactivated in vivo to form an electrophilic intermediate(s). Results from this work demonstrate the utility of using horseradish peroxidase and hydrogen peroxide (HRP/P) for assessing the skin sensitization potential of pro-haptens. In comparison with "direct" reactivity assessments without HRP/P, statistically significant increases in peptide depletion for all pro-haptens examined were observed following coincubation with HRP/P. Conversely, the percent peptide depletion for all pre-haptens was equally high (> 40% depletion) with and without HRP/P demonstrating an auto-oxidation pathway. In contrast, peptide depletion for all nonsensitizing chemicals examined was low with and without HRP/P. The optimal HRP/P concentrations, incubation time and optimal peptide:chemical ratio were determined using a sensitive and selective high-performance liquid chromatography tandem mass spectrometry detection method. Dithiothreitol was incorporated to reverse the dimerization of the thiol-containing cysteine peptide nucleophile. This preliminary work shows the potential to incorporate an enzyme-mediated activation step for pro-haptens into an in chemico skin sensitization assay that results in the detection of all types of sensitizers.

Effects of Methyl Mercury in Combination with Polychlorinated Biphenyls and Brominated Flame Retardants on the Uptake of Glutamate in Rat Brain Synaptosomes: A Mathematical Approach for the Study of Mixtures

Stavenes Andersen, I., Voie, O. A., Fonnum, F., Mariussen, E. — Tue, 27 Oct 2009 12:53:45 PDT

Regulatory limit values for toxicants are in general determined by the toxicology of the single compounds. However, little is known about their combined effects. Methyl mercury (MeHg), polychlorinated biphenyls (PCBs), and brominated flame retardants (BFRs) are dominant contaminants in the environment and food. MeHg is a well known neurotoxicant, especially affecting the developing brain. There is increasing evidence that PCB and BFRs also have neurotoxic effects. An enhanced effect of these toxicants, due to either synergistic or additive effects, would be considered as a risk for the fetal development. Here we studied the combinatorial effects of MeHg in combination with PCB or BFR on the reuptake of glutamate in synaptosomes. To provide the optimal conclusion regarding type of interaction, we have analyzed the data using two mathematical models, the Löewe model of additivity and Bliss’ model of independent action. Binary and ternary mixtures in different proportions were made. The toxicants had primarily additive effects, as shown with both models, although tendencies towards synergism were observed. MeHg was by far the most potent inhibitor of uptake with an EC₅₀ value of 0.33µM. A reconstituted mixture from a relevant fish sample was made in order to elucidate which chemical was responsible for the observed effect. Some interaction was experienced between PCB and MeHg, but in general MeHg seemed to explain the observed effect. We also show that mixture effects should not be assessed by effect addition.

Long-term Cognitive Impairments in Adult Rats Treated Neonatally with {beta}-N-Methylamino-L-Alanine

Karlsson, O., Roman, E., Brittebo, E. B. — Tue, 27 Oct 2009 12:53:45 PDT

Most cyanobacteria (blue-green algae) can produce the neurotoxin β-N-methylamino-L-alanine (BMAA). Dietary exposure to BMAA has been suggested to be involved in the etiology of the neurodegenerative disease amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC). Little is known about BMAA-induced neurotoxicity following neonatal administration. Our previous studies have revealed an uptake of BMAA in the hippocampus and striatum of neonatal mice. Furthermore, rats treated with BMAA during the neonatal period displayed acute but transient motoric disturbances and failed to show habituation at juvenile age suggesting impairments in learning functions. In the present study, the aim was to investigate long-term behavioral effects of BMAA administration in neonatal rats. BMAA was administered on postnatal days 9–10 (200 or 600 mg/kg; subcutaneous injection). Spatial learning and memory was investigated in adulthood using the radial arm maze test. The results revealed impaired learning but not memory in BMAA-treated animals. The observed impairments were not due to alterations in motoric capacity, general activity, or behavioral profiles, as assessed in the multivariate concentric square field (MCSF) and open field tests. An aversive stimulus in the MCSF test revealed impairments in avoidance learning and/or memory. There was no difference in basal serum corticosterone levels in BMAA-treated animals, indicating that the observed long-term effects were not secondary to an altered basal hypothalamic-pituitary-adrenal axis function. The present data demonstrated long-term learning impairments following neonatal BMAA administration. Further studies on biochemical effects in various brain regions and subsequent behavioral alterations are needed to elucidate the mechanisms of BMAA-induced developmental neurotoxicity.

Investigations of the Use of Bioavailability Data to Adjust Occupational Exposure Limits for Active Pharmaceutical Ingredients

Tue, 27 Oct 2009 12:53:45 PDT

Occupational exposure limits (OELs) for active pharmaceutical ingredients have traditionally been established using no-observed-adverse-effect levels derived from clinical studies employing po and iv routes of administration and by applying default uncertainty factors or chemical-specific adjustment factors. However, exposure by the inhalation or dermal route is more relevant in terms of occupational safety. In this investigation, to explore new methods for route-to-route extrapolation, the bioavailability of MK-0679, a leukotriene D₄ receptor antagonist, was compared following iv, po, intranasal (in), or intratracheal (it) administration. The relative bioavailability of MK-0679 was iv it > po in. Bioavailability correction factors (BCFs) of 2.0 and 0.6 were derived from these data to adjust a hypothetical OEL of 0.1 mg/m³ for MK-0679 with particle sizes of 10 and 50 µm, respectively. These BCFs were used to adjust the OEL established using po clinical data, to reflect the differences in bioavailability following deposition in different regions of the respiratory tract. To further investigate how bioavailability data could be used in setting OELs, a preliminary pharmacokinetic (PK) model was developed to describe the time course of plasma concentrations using the data from the route comparison study. An inhalation study was then performed to test the validity of using either empirical data or modeling approaches to derive BCFs when setting OELs. These investigations demonstrated how the use of route-specific PK data could reduce some of the uncertainties associated with route-to-route extrapolation and allow for improved precision and quantitative adjustments when establishing OELs. Further investigations are needed to better understand the factors responsible for differences in systemic uptake following deposition in different regions of the respiratory tract and how these can be generalized across different classes of soluble compounds.

N-acetyltransferase 2 Genotype Modification of Active Cigarette Smoking on Breast Cancer Risk among Hispanic and Non-Hispanic White Women

Baumgartner, K. B., Schlierf, T. J., Yang, D., Doll, M. A., Hein, D. W. — Tue, 27 Oct 2009 12:53:45 PDT

While it has been demonstrated that cigarette smoke contains aromatic and heterocyclic amines that initiate carcinogenesis, the association between cigarette smoking and breast cancer remains controversial. N-acetyltransferase 2 (NAT2) catalyzes arylamine carcinogen biotransformation and NAT2 genetic polymorphisms may contribute to differential susceptibility to breast cancer. We tested whether NAT2 modified the association between cigarette smoking and breast cancer risk in a population-based study of Hispanic and non-Hispanic white women in the Southwest United States. Data were available for cigarette smoking and NAT2 polymorphisms for 717 cases (Hispanic, 251 and non-Hispanic white, 466) and 735 controls (Hispanic, 245 and non-Hispanic white, 490). NAT2 genotypes were translated into rapid, intermediate, slow, or very slow acetylator phenotypes. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for the joint association of NAT2 with smoking on breast cancer risk were estimated using logistic regression. Non-Hispanic white women were more likely (p < 0.001) than Hispanic women to have a slow (41.7 vs. 33.5%) or very slow (19.0 vs. 11.1%) acetylator status and less likely to have rapid/intermediate phenotypes (39.2 vs. 54.4%). Breast cancer risk was significantly increased in non-Hispanic white women with a very slow acetylator phenotype who smoked: ever versus never (OR, 2.57; 95% CI, 1.49–4.41), never versus former (OR, 2.69; 95% CI, 1.41–5.17) or current (OR, 2.46; 95% CI, 1.07–5.65), and 16 + pack-years (OR, 2.29; 95% CI, 1.16–4.51). Results for Hispanic women were not statistically significant. These findings support smoking as a risk factor for breast cancer among non-Hispanic white women with very slow NAT2 acetylator phenotype.

Automated Dose-Response Analysis of the Relative Hepatic Gene Expression Potency of TCDF in C57BL/6 Mice

Tue, 27 Oct 2009 12:53:45 PDT

Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF), relative to TCDD with complementary histopathology, TCDD and TCDF tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay data. Immature ovariectomized C57BL/6 mice were gavaged with 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 µg/kg TCDD, the World Health Organization TEF-adjusted doses (10 x TCDD dose) of TCDF (0.3, 1, 3, 10, 30, 100, or 300 µg/kg), or sesame oil vehicle and killed at 72 h. Two thousand two hundred eighty-eight and 1347 genes were differentially expressed (P1(t) > 0.90) at one or more doses by TCDD and TCDF, respectively. Automated dose-response modeling (ToxResponse Modeler) identified a total of 1027 and 837 genes with either a sigmoidal, exponential, linear, Gaussian, or quadratic dose-response relationship 72 h after treatment in TCDD and TCDF, respectively. Two hundred seventy genes exhibited a sigmoidal TCDD-induced dose-response (ED_50s from 0.08 to 42.2 µg/kg) compared to only 179 sigmoidal responsive genes (ED_50s from 0.74 to 299.9 µg/kg) elicited by TCDF. Of the 1027 TCDD dose-responsive genes, 654 were not examined further due to the lack of a dose response elicited by TCDF. Of the 373 genes that exhibited a TCDD and TCDF dose response, REPs were calculated for the 83 genes that exhibited comparable sigmoidal curve shapes and slopes. The median REP for these 83 genes was 0.10, with a maximum REP of 0.56 and a minimum of 0.01. REPs of 0.04 were also calculated for EROD and increase in relative liver weight (RLW) at 72 h. Collectively, the lower number of TCDF-induced genes compared to TCDD and the 0.04 REPs for EROD activity and increased RLW are not consistent with the TEF of 0.10 for the hepatotoxicity of TCDF in C57BL/6 mice at 72 h.

Comparative Analysis of AhR-Mediated TCDD-Elicited Gene Expression in Human Liver Adult Stem Cells

Kim, S., Dere, E., Burgoon, L. D., Chang, C.-C., Zacharewski, T. R. — Tue, 27 Oct 2009 12:53:45 PDT

Time course and dose-response studies were conducted in HL1-1 cells, a human liver cell line with stem cell–like characteristics, to assess the differential gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compared with other established models. Cells were treated with 0.001, 0.01, 0.1, 1, 10, or 100nM TCDD or dimethyl sulfoxide vehicle control for 12 h for the dose-response study, or with 10nM TCDD or vehicle for 1, 2, 4, 8, 12, 24, or 48 h for the time course study. Elicited changes were monitored using a human cDNA microarray with 6995 represented genes. Empirical Bayes analysis identified 144 genes differentially expressed at one or more time points following treatment. Most genes exhibited dose-dependent responses including CYP1A1, CYP1B1, ALDH1A3, and SLC7A5 genes. Comparative analysis of HL1-1 differential gene expression to human HepG2 data identified 74 genes with comparable temporal expression profiles including 12 putative primary responses. HL1-1–specific changes were related to lipid metabolism and immune responses, consistent with effects elicited in vivo. Furthermore, comparative analysis of HL1-1 cells with mouse Hepa1c1c7 hepatoma cell lines and C57BL/6 hepatic tissue identified 18 and 32 commonly regulated orthologous genes, respectively, with functions associated with signal transduction, transcriptional regulation, metabolism and transport. Although some common pathways are affected, the results suggest that TCDD elicits species- and model-specific gene expression profiles.

Dioxin-Dependent and Dioxin-Independent Gene Batteries: Comparison of Liver and Kidney in AHR-Null Mice

Boutros, P. C., Bielefeld, K. A., Pohjanvirta, R., Harper, P. A. — Tue, 27 Oct 2009 12:53:46 PDT

The aryl hydrocarbon receptor (AHR) is a widely expressed ligand-dependent transcription factor that mediates cellular responses to dioxins and other planar aromatic hydrocarbons. Ahr-null mice are refractory to the toxic effects of dioxin exposure. Although some mechanistic aspects of AHR activity are well understood, the tissue specificity of AHR effects remains unclear, both during development and following administration of exogenous ligands. To address the latter issue, we defined and compared transcriptional responses to dioxin exposure in the liver and kidney of wild-type and Ahr-null adult C57BL/6J mice treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin or corn-oil vehicle. In both tissues, essentially all effects of dioxin on hepatic mRNA levels were mediated by the AHR. Although 297 genes were altered by dioxin exposure in the liver, only 17 were changed in the kidney, including a number of well-established AHR target genes. Ahr genotype had a large effect in both tissues, profoundly remodeling both the renal and hepatic transcriptomes. Surprisingly, a large number of genes were affected by Ahr genotype in both tissues, suggesting the presence of a basal AHR gene battery. Alterations of the renal transcriptome in Ahr-null animals were associated with perturbation of specific functional pathways and enrichment of specific DNA motifs. Our results demonstrate the importance of intertissue comparisons, highlight the basal role of the AHR in liver and kidney, and support a role in development or normal physiology.

Divergent Transcriptomic Responses to Aryl Hydrocarbon Receptor Agonists between Rat and Human Primary Hepatocytes

Tue, 27 Oct 2009 12:53:46 PDT

Toxicogenomics has great potential for enhancing our understanding of environmental chemical toxicity, hopefully leading to better informed human health risk assessments. This study employed toxicogenomic technology to reveal species differences in response to two prototypical aryl hydrocarbon receptor (AHR) agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin and the polychlorinated biphenyl (PCB) congener PCB 126. Dose-responses of primary cultures of rat and human hepatocytes were determined using species-specific microarrays sharing over 4000 gene orthologs. Forty-seven human and 79 rat genes satisfied dose-response criteria for both chemicals and were subjected to further analysis including the calculation of the 50% effective concentration and the relative potency (REP) of PCB 126 for each gene. Only five responsive orthologous genes were shared between the two species; yet, the geometric mean of the REPs for all rat and human modeled responsive genes were 0.06 (95% confidence interval [CI]; 0.03–0.1) and 0.002 (95% CI; 0.001–0.005), respectively, suggesting broad species differences in the initial events that follow AHR activation but precede toxicity. This indicates that there are species differences in both the specific genes that responded and the agonist potency and REP for those genes. This observed insensitivity of human cells to PCB 126 is consistent with more traditional measurements of AHR activation (i.e., cytochrome P450 1A1 enzyme activity) and suggests that the species difference in PCB 126 sensitivity is likely due to certain aspects of AHR function. That a species divergence also exists in this expanded AHR-regulated gene repertoire is a novel finding and should help when extrapolating animal data to humans.

Editorial Board

Fri, 11 Sep 2009 17:40:57 PDT

SUBSCRIPTIONS

Fri, 11 Sep 2009 17:40:57 PDT

Cross-talk between Transcription Factors AhR and Nrf2: Lessons for Cancer Chemoprevention from Dioxin

Hayes, J. D., Dinkova-Kostova, A. T., McMahon, M. — Fri, 11 Sep 2009 17:40:57 PDT

Cellular Stress Response Pathway System as a Sentinel Ensemble in Toxicological Screening

Simmons, S. O., Fan, C.-Y., Ramabhadran, R. — Fri, 11 Sep 2009 17:40:57 PDT

High costs, long test times, and societal concerns related to animal use have required the development of in vitro assays for the rapid and cost-effective toxicological evaluation and characterization of compounds in both the pharmaceutical and environmental arenas. Although the pharmaceutical industry has developed very effective, high-throughput in vitro assays for determining the therapeutic potential of compounds, the application of this approach to toxicological screening has been limited. A primary reason for this is that while drug candidate screens are directed to a specific target/mechanism, xenobiotics can cause toxicity through any of a myriad of undefined interactions with cellular components and processes. Given that it is not practical to design assays that can interrogate each potential toxicological target, an integrative approach is required if there is to be a rapid and low-cost toxicological evaluation of chemicals. Cellular stress response pathways offer a viable solution to the creation of a set of integrative assays as there is a limited and hence manageable set (a small ensemble of 10 or less) of major cellular stress response pathways through which cells mount a homoeostatic response to toxicants and which also participate in cell fate/death decisions. Further, over the past decades, these pathways have been well characterized at a molecular level thereby enabling the development of high-throughput cell-based assays using the components of the pathways. Utilization of the set of cellular stress response pathway-based assays as indicators of toxic interactions of chemicals with basic cellular machinery will potentially permit the clustering of chemicals based on biological response profiles of common mode of action (MOA) and also the inference of the specific MOA of a toxicant. This article reviews the biochemical characteristics of the stress response pathways, their common architecture that enables rapid activation during stress, their participation in cell fate decisions, the essential nature of these pathways to the organism, and the biochemical basis of their cross-talk that permits an assay ensemble screening approach. Subsequent sections describe how the stress pathway ensemble assay approach could be applied to screening potentially toxic compounds and discuss how this approach may be used to derive toxicant MOA from the biological activity profiles that the ensemble strategy provides. The article concludes with a review of the application of the stress assay concept to noninvasive in vivo assessments of chemical toxicants.

Biologically Relevant Exposure Science for 21st Century Toxicity Testing

Hubal, E. A. C. — Fri, 11 Sep 2009 17:40:57 PDT

High visibility efforts in toxicity testing and computational toxicology including the recent National Research Council of the National Academies (NRC) report, Toxicity Testing in the 21st Century: A Vision and Strategy (NRC, 2007a), raise important research questions and opportunities for the field of exposure science. The authors of the National Academies report (NRC, 2007a) emphasize that population-based data and human exposure information are required at each step of their vision for toxicity testing and that these data will continue to play a critical role in both guiding development and use of the toxicity information. In fact, state-of-the-art exposure science is essential for translation of toxicity data to assess potential for risk to individuals and populations and to inform public health decisions. As we move forward to implement the NRC vision, a transformational change in exposure science is required. Application of a fresh perspective and novel techniques to capture critical determinants at biologically motivated resolution for translation from controlled in vitro systems to the open multifactorial system of real-world human-environment interaction will be critical. Development of an exposure ontology and knowledgebase will facilitate extension of network analysis to the individual and population for translating toxicity information and assessing health risk. Such a sea change in exposure science is required to incorporate consideration of lifestage, genetic susceptibility, and interaction of nonchemical stressors for holistic assessment of risk factors associated with complex environmental disease. A new generation of scientific tools has emerged to rapidly measure signals from cells, tissues, and organisms following exposure to chemicals. Investment in 21st century exposure science is now required to fully realize the potential of the NRC vision for toxicity testing.

Are In Vitro Tests Suitable for Regulatory Use?

Hartung, T., Daston, G. — Fri, 11 Sep 2009 17:40:57 PDT

"All models are wrong, some models are useful"

George E.P. Box, "Robustness in the strategy of scientific model building", 1979. This quote from George Box was the title of a book chapter on mathematical models—exactly the type of models which we will not address here, although a lot of the general reasoning can as well be translated to them, especially because they depend on the input and thus on the limitations of either in vitro or in vivo data, when applied for regulatory toxicology. This article was prompted by the 2008 SOT/EuroTox debate (March 2008 in Seattle and October 2008 in Rhodes), which challenged us in an Hegelian approach as thesis and antithesis to present with changing roles on both occasions on the statement "In vitro tests are useless for regulatory use." Here, we would like following Hegel a summary of thesis and antithesis, but also try to outline the synthesis.

Introducing the "TCDD-Inducible AhR-Nrf2 Gene Battery"

Yeager, R. L., Reisman, S. A., Aleksunes, L. M., Klaassen, C. D. — Fri, 11 Sep 2009 17:40:57 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 µg/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid–synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice.

Effect of Dietary Selenium and Cigarette Smoke on Pulmonary Cell Proliferation in Mice

Fri, 11 Sep 2009 17:40:57 PDT

The objective of this study was to determine if dietary selenium could inhibit pulmonary cell proliferation in control and cigarette smoke-exposed female A/J mice. Selenium in the form of sodium selenite was supplemented to purified diets similar to the AIN-93M diet to yield 0.15, 0.5, or 2.0 mg selenium/kg diet. After 3 weeks, mice in each dietary group were divided into two subgroups; one used as control, whereas the other was exposed to cigarette smoke for five consecutive days. Mice from both groups were euthanized 3 days later. Mice were administered bromodeoxyuridine in the drinking water starting 5 days before the initiation of the smoke exposure and continuing until they were euthanized. After euthanasia, the left lung lobe was processed for histology and cell proliferation analysis. Cigarette smoke increased cell proliferation in the terminal bronchioles and large airways, but not in alveoli. High-selenium diets inhibited cell proliferation in the alveoli, terminal bronchioles and large airways areas in both control and smoke-exposed mice. Increasing the dietary selenium level led to increased selenium levels in the blood and lung, and increased glutathione peroxidase (GPx) activity in the lung. Cytochrome P-450 1A1 protein levels in the lung were increased by cigarette smoke but were not affected by dietary selenium. It is concluded that dietary selenium inhibits pulmonary cell proliferation in both control and cigarette smoke-exposed mice, indicating that selenium is inhibiting cell proliferation independently of smoke exposure, and that this inhibition may be related to selenium concentration and GPx activity in the lung.

Dioxin Increases the Interaction Between Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha at Human Promoters

Ahmed, S., Valen, E., Sandelin, A., Matthews, J. — Fri, 11 Sep 2009 17:40:57 PDT

Recent studies have shown that activated aryl hydrocarbon receptor (AHR) induced the recruitment of estrogen receptor- (ER) to AHR-regulated genes and that AHR is recruited to ER-regulated genes. However, these findings were limited to a small number of well-characterized AHR- or ER-responsive genes with little knowledge of what was occurring at other genomic regions. In this study, we showed using chromatin immunoprecipitation followed by hybridization to promoter focused microarrays (ChIP-chip) that 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment significantly increased the overlap of genomic regions bound by both AHR and ER. Conventional and sequential ChIPs confirmed the recruitment of AHR and ER to many of the identified regions. Transcription factor binding site analysis revealed an overrepresentation of aryl hydrocarbon receptor response elements in regions bound by both AHR and ER, suggesting that AHR was the important factor determining the recruitment of ER to these regions. RNA interference-mediated knockdown of AHR confirmed its requirement for the recruitment of ER to some, but not all, of the shared regions. Our findings demonstrate not only that dioxin induces the recruitment of ER to AHR target genes but also that AHR is recruited to estrogen-responsive regions in a gene-specific manner, suggesting that AHR utilizes both of these mechanisms to modulate estrogen-dependent signaling.

Roles of the Genetic Polymorphisms of Alcohol-Metabolizing Enzymes on the Immunology in High-Risk Drinkers

Fri, 11 Sep 2009 17:40:57 PDT

Alcohol metabolism involves several enzymes and the individual genetic variations in the alcohol metabolism are related to the absorption, distribution, and elimination of alcohol and metabolites such as acetaldehyde. Therefore, the genetic variations of alcohol-metabolizing enzymes are responsible for the different toxicity of alcohol in several organs like liver and immunological systems. The purpose of this study was to evaluate if the life styles such as drinking and smoking and the genetic variations of alcohol-metabolizing enzymes (ADH2, ALDH2, CYP2E1, and CAT) were associated with the immunological biomarkers. In this study, 105 high-risk drinkers and 102 low-risk drinkers who were excluded from the immune-related diseases and other critical diseases were enrolled to evaluate the immunological functions. Counts of white blood cells, mononuclear cells, and lymphocyte subpopulations, and liver and immunological function tests were measured. Genotypes of alcohol-metabolizing enzymes were assayed by a real-time PCR and PCR-restriction fragment length polymorphism. Generally, the activity of aspartate aminotransferase (AST) was higher than that of alanine aminotransferase (ALT) in alcoholics; however, the activities of AST and ALT were simultaneously elevated in general hepatitis except for alcohol-induced hepatitis. Thus, the higher ratio of AST/ALT was used to be a marker for the alcohol-induced abnormal liver function. Glutamyltransferase (GGT) is produced by the liver cell microsomes and is a useful laboratory marker as an indicator of early liver cell damage. An increase in GGT concentration has been regarded as a marker of alcohol consumption or liver disease. In addition, the synergistic effects of smoking and drinking on the count of white blood cell (WBC) and mononuclear cells were found to be significant. Furthermore, there were higher OR to become high-risk drinkers in subjects with the combination of ALDH2 (*1/*1) genotype and either genotype of ADH2 or CYP2E1 than the others with other combinations of genotypes. Additionally, there were more abnormal immunological tests in the subjects with higher activity of ADH2 and lower activity of ALDH2. Our results suggested that the habits of drinking, smoking, and betel chewing, and genetic variations of alcohol metabolism were associated with the immunological biomarkers.

Induction of Suppressors of Cytokine Signaling by the Trichothecene Deoxynivalenol in the Mouse

Amuzie, C. J., Shinozuka, J., Pestka, J. J. — Fri, 11 Sep 2009 17:40:57 PDT

Deoxynivalenol (DON), a trichothecene mycotoxin found in grains and cereal–based foods worldwide, impairs weight gain in experimental animals but the underlying mechanisms remain undetermined. Oral exposure to DON induces rapid and transient upregulation of proinflammatory cytokine expression in the mouse. The latter are known to induce several suppressors of cytokine signaling (SOCS), some of which impair growth hormone (GH) signaling. We hypothesized that oral exposure to DON will induce SOCS expression in the mouse. Real-time PCR and cytokine bead array revealed that oral gavage with DON rapidly (1 h) induced tumor necrosis factor- and interleukin-6 mRNA and protein expression in several organs and plasma, respectively. Upregulation of mRNAs for four well-characterized SOCS (CIS [cytokine-inducible SH2 domain protein], SOCS1, SOCS2, and SOCS3) was either concurrent with (1 h) or subsequent to cytokine upregulation (2 h). Notably, DON-induced SOCS3 mRNAs in muscle, spleen and liver, with CIS1, SOCS1, and SOCS2 occurring to a lesser extent. Hepatic SOCS3 mRNA was a very sensitive indicator of DON exposure with SOCS3 protein being detectable in the liver well after the onset of cytokine decline (5 h). Furthermore, hepatic SOCS upregulation was associated with about 75% suppression of GH-inducible insulin-like growth factor acid labile subunit. Taken together, DON-induced cytokine upregulation corresponded to increased expression of several SOCS, and was associated with suppression of GH-inducible gene expression in the liver.

Trovafloxacin Enhances TNF-Induced Inflammatory Stress and Cell Death Signaling and Reduces TNF Clearance in a Murine Model of Idiosyncratic Hepatotoxicity

Fri, 11 Sep 2009 17:40:57 PDT

Therapy employing the fluoroquinolone antibiotic, trovafloxacin (TVX) was curtailed due to idiosyncratic hepatotoxicity. Previous studies in mice showed that a nonhepatotoxic inflammatory stress induced by tumor necrosis factor (TNF) synergized with a nonhepatotoxic dose of TVX to cause liver injury. The purpose of this study was to explore mechanisms by which TVX interacts with TNF to cause liver injury. TVX pretreatment prolonged the peak of plasma TNF after its administration. This prolongation of TNF by TVX was critical to the development of hepatotoxicity. The prolongation of TNF concentration in plasma was primarily due to reduced clearance when compared with secondary biosynthesis. TNF is cleared from plasma by binding to soluble TNF receptors (TNFRs) which are eliminated by the kidney; however, the plasma concentrations of soluble TNFRs were not reduced, and biomarkers of renal dysfunction were not elevated in TVX/TNF-treated mice. Two injections of TNF mimicked the prolongation of the TNF peak by TVX and caused liver injury, but injury was less severe than after TVX/TNF coexposure. TVX enhanced the induction of proinflammatory cytokines by TNF. Additionally, TVX sensitized Hepa1c1c7 cells to TNF-induced killing in a concentration-dependent manner and increased both potency and efficacy of TNF to activate effector caspases that were critically involved in cell death from TVX/TNF coexposure. In summary, TVX reduced the clearance of TNF independent of either receptor shedding or kidney dysfunction. Additionally, TVX interacted with TNF to enhance inflammation and sensitize hepatocytes to TNF-induced cell death.

Halothane-Induced Liver Injury is Mediated by Interleukin-17 in Mice

Kobayashi, E., Kobayashi, M., Tsuneyama, K., Fukami, T., Nakajima, M., Yokoi, T. — Fri, 11 Sep 2009 17:40:57 PDT

Drug-induced liver injury is a major problem in drug development and clinical drug therapy. In most cases the mechanisms are still unknown, thus, it is difficult to predict or prevent these reactions. It has been known that halothane, an inhaled anesthetic, induces liver injury. To investigate the mechanisms of halothane-induced liver injury, we used a recently established mouse model of liver injury. The expression of transcription factors and cytokines specific for Th1 and Th2 (helper T cells), respectively, were compared between BALB/c and C57BL/6 mice. The mRNA expression ratios of mouse T-bet(a Th1-specific transcription factor)/GATA-binding protein (GATA-3, a Th2-specific transcription factor) and interferon /interleukin (IL)-10 were lower in BALB/c mice compared with C57BL/6 mice, suggesting that a typical Th1 or Th2-dominant response could not be distinguished in halothane-induced liver injury. We observed increases of the plasma IL-17 level and hepatic macrophage inflammatory protein 2 expression in halothane-administrated BALB/c mice, as well as neutrophil infiltration. Neutralization of IL-17 suppressed the hepatotoxic effect of halothane. Administration of recombinant IL-17 (1 µg per mouse, single ip) to the halothane-treated mice resulted in a remarkable increase of alanine and aspartate aminotransferases. In conclusion, we demonstrated that IL-17 is involved in the halothane-induced liver injury.

Effects of 18 Perfluoroalkyl Compounds on mRNA Expression in Chicken Embryo Hepatocyte Cultures

Hickey, N. J., Crump, D., Jones, S. P., Kennedy, S. W. — Fri, 11 Sep 2009 17:40:57 PDT

Many studies have characterized the effects of perfluoroalkyl compounds (PFCs) in mammalian species, but limited information exists on the effects of PFCs in birds. PFCs have been detected in serum and liver of avian wildlife worldwide. While the molecular mechanisms have yet to be elucidated in detail, PFCs alter lipid metabolism through peroxisome proliferation, xenobiotic metabolism by activating the cytochrome P450 (CYP) system, and serum cholesterol levels by inducing or repressing key genes. Here, we employed a simple messenger RNA (mRNA) screening method using quantitative PCR to assess the effects of PFCs on mRNA expression in chicken embryo hepatocytes (CEH). CEH cultures were treated with perfluoroalkyl sulfonates and perfluoroalkyl carboxylates of varying chain lengths and linear or technical grade potassium perfluoro-1-octane sulfonate (L-PFOS and T-PFOS). T-PFOS comprised 80% perfluorooctane sulfonate isomers (62% linear) and various PFCs and inorganic salts. Relative mRNA expression levels of the following genes were examined: acyl-CoA oxidase (ACOX), liver fatty acid–binding protein (L-FABP), CYP1A4/1A5 and CYP4B1, 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase, and sterol regulatory element–binding protein 2 (SREBP2). Compared to L-PFOS, T-PFOS altered the mRNA expression level of more genes and produced greater fold changes. L-FABP was upregulated by PFCs greater than or equal to eight carbons, while CYPs were upregulated by PFCs less than or equal to eight carbons. ACOX, HMG-CoA, and SREBP2 showed little to no change following PFC exposure. This study is the first to expose CEH cultures to multiple PFCs in vitro and demonstrates that exposure to PFC solutions of different isomeric content or chain length causes variable transcriptional responses.

MRI Characterization of Structural Mouse Brain Changes in Response to Chronic Exposure to the Glufosinate Ammonium Herbicide

Fri, 11 Sep 2009 17:40:57 PDT

Glufosinate ammonium (GLA) is the active component of herbicides widely used in agriculture, truck farming, or public domains. GLA acts by inhibiting the plant glutamine synthetase (GlnS). It also inhibits mammalian GlnS in vitro and ex vivo. In the central nervous system this enzyme is exclusively localized in glial cells. Whereas acute neurotoxic effects of GLA are well documented, long-term effects during chronic exposure at low doses remain largely undisclosed. In the present work, C57BL/6J mice were treated intraperitoneally with 2.5, 5, and 10 mg/kg of GLA three times a week during 10 weeks. Cerebral magnetic resonance imaging (MRI) experiments were performed at high field (9.4 T) and the images were analyzed with four texture analysis (TA) methods. TA highlighted structural changes in seven brain structures after chronic GLA treatments. Changes are dose dependent and can be seen at a dose as low as 2.5 mg/kg for two areas, namely hippocampus and somatosensorial cortex. Glial fibrillary acidic protein (GFAP) expression in the same seven brain structures and GlnS activity in the hippocampus and cortex areas were also studied. The number of GFAP-positive cells is modified in six out of the seven areas examined. GlnS activity was significantly increased in the hippocampus but not in the cortex. These results indicate some kind of suffering at the cerebral level after chronic GLA treatment. Changes in TA were compared with the modification of the number of GFAP-positive astrocytes in the studied brain areas after GLA treatment. We show that the noninvasive MRI-TA is a sensitive method and we suggest that it would be a very helpful tool that can efficiently contribute to the detection of cerebral alterations in vivo during chronic exposure to xenobiotics.

Iron-Induced Oxidative Injury Differentially Regulates PI3K/Akt/GSK3{beta} Pathway in Synaptic Endings from Adult and Aged Rats

Uranga, R. M., Giusto, N. M., Salvador, G. A. — Fri, 11 Sep 2009 17:40:58 PDT

In this work we study the state of phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK3β) signaling during oxidative injury triggered by free iron using cerebral cortex synaptic endings isolated from adult (4-month-old) and aged (28-month-old) rats. Synaptosomes were exposed to FeSO₄ (50µM) for different periods of time and synaptosomal viability and the state of the PI3K/Akt/GSK3β pathway were evaluated in adult and aged animals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and lactate dehydrogenase leakage were significantly affected in both age groups. However, aged animals showed a greater susceptibility to oxidative stress. In adults, Akt was activated after a brief exposure time (5 min), whereas in aged animals activation occurred after 5 and 30 min of incubation with the metal ion. GSK3β phosphorylation showed the same activation pattern as that observed for Akt. Both Akt and GSK3β phosphorylation were dependent on PI3K activation. Extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation was temporally coincident with Akt activation and was PI3K dependent in adults, whereas ERK1/2 activation in aged rats was higher than that observed in adults and showed no dependence on PI3K activity. We demonstrate here that synaptic endings from adult and aged animals subjected to iron-induced neurotoxicity show a differential profile in the activation of PI3K/Akt/GSK3β. Our results strongly suggest that the increased susceptibility of aged animals to oxidative injury provokes a differential modulation of key signaling pathways involved in synaptic plasticity and neuronal survival.

Caenorhabditis elegans Metallothioneins Protect against Toxicity Induced by Depleted Uranium

Fri, 11 Sep 2009 17:40:58 PDT

Depleted uranium (DU) is a dense and heavy metal used in armor, ammunition, radiation shielding, and counterbalances. The military usage has led to growing public concern regarding the health effects of DU. In this study, we used the nematode, Caenorhabditis elegans, to evaluate the toxicity of DU and its effects in knockout strains of metallothioneins (MTs), which are small thiol-rich proteins that have numerous functions, such as metal sequestration, transport, and detoxification. We examined nematode viability, the accumulation of uranium, changes in MT gene expression by quantitative reverse transcription-PCR, and the induction of green fluorescent protein under the control of the MT promoters, following exposure to DU. Our results indicate that (1) DU causes toxicity in a dose-dependent manner; (2) MTs are protective against DU exposure; and (3) nematode death by DU is not solely a reflection of intracellular uranium concentration. (4) Furthermore, only one of the isoforms of MTs, metallothionein-1 (mtl-1), appears to be important for uranium accumulation in C. elegans. These findings suggest that these highly homologous proteins may have subtle functional differences and indicate that MTs mediate the response to DU.

A Minimally Invasive, Translational Biomarker of Ketamine-Induced Neuronal Death in Rats: microPET Imaging Using 18F-Annexin V

Fri, 11 Sep 2009 17:40:58 PDT

It has been reported that suppression of N-methyl-D-aspartate (NMDA) receptor function by ketamine may trigger apoptosis of neurons when given repeatedly during the brain growth spurt period. Because microPET scans can provide in vivo molecular imaging at sufficient resolution, it has been proposed as a minimally invasive method for detecting apoptosis using the tracer ¹⁸F-labeled annexin V. In this study, the effect of ketamine on the metabolism and integrity of the rat brain were evaluated by investigating the uptake and retention of ¹⁸F-fluorodeoxyglucose (FDG) and ¹⁸F-annexin V using microPET imaging. On postnatal day (PND) 7, rat pups in the experimental group were exposed to six injections of ketamine (20 mg/kg at 2-h intervals) and control rat pups received six injections of saline. On PND 35, 37 MBq (1 mCi) of ¹⁸F-FDG or ¹⁸F-annexin V was injected into the tail vein of treated and control rats, and static microPET images were obtained over 1 (FDG) and 2 h (annexin V) following the injection. No significant difference was found in ¹⁸F-FDG uptake in the regions of interest (ROIs) in the brains of ketamine-treated rats compared with saline-treated controls. The uptake of ¹⁸F-annexin V, however, was significantly increased in the ROI of ketamine-treated rats. Additionally, the duration of annexin V tracer washout was prolonged in the ketamine-treated animals. These results demonstrate that microPET imaging is capable of distinguishing differences in retention of ¹⁸F-annexin V in different brain regions and suggests that this approach may provide a minimally invasive biomarker of neuronal apoptosis in rats.

Toluene-Induced Hearing Loss in the Guinea Pig

Fri, 11 Sep 2009 17:40:58 PDT

Toluene is a high-production industrial solvent, which can disrupt the auditory system in rats. However, toluene-induced hearing loss is species dependent. For instance, despite long-lasting exposures to high concentrations of aromatic solvent, no study has yet succeeded in causing convincing hearing loss in the guinea pig. This latter species can be characterized by two metabolic particularities: a high amount of hepatic cytochrome P-450s (P-450s) and a high concentration of glutathione in the cochlea. It is therefore likely that the efficiency of both the hepatic and cochlear metabolisms plays a key role in the innocuousness of the hearing of guinea pigs to exposure to solvent. The present study was carried out to test the auditory resistance to toluene in glutathione-depleted guinea pigs whose the P-450 activity was partly inhibited. To this end, animals on a low-protein diet received a general P-450 inhibitor, namely SKF525-A. Meanwhile, they were exposed to 1750 ppm toluene for 4 weeks, 5 days/week, 6 h/day. Auditory function was tested by electrocochleography and completed by histological analyses. For the first time, a significant toluene-induced hearing loss was provoked in the P-450–inhibited guinea pigs. However, the ototoxic process caused by the solvent exposure was different from that observed in the rat. Only the stria vascularis and the spiral fibers were disrupted in the apical coil of the cochlea. The protective mechanisms developed by guinea pigs are discussed in the present publication.

Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism

Lehraiki, A., Racine, C., Krust, A., Habert, R., Levacher, C. — Fri, 11 Sep 2009 17:40:58 PDT

Data from experiments conducted almost exclusively in the rat have established that some phthalates have deleterious effects on the fetal testis probably due to their antiandrogenic and/or estrogenic effects, but their mechanisms of action remain unknown. A recent study reported that phthalates also have deleterious effects on human fetal testis with germ cell number, but not steroidogenesis altered. Therefore, we used organ culture of fetal testes at different stages of development to analyze the direct effects of phthalates on both steroidogenesis and gonocyte development and to determine if the effects of MEHP on these functions reported in the rat can be extended to other mammalian species. We defined specific periods of sensitivity of the fetal mouse testis to MEHP for these two functions and showed that the effects of phthalates on steroidogenesis vary with the developmental stage. Conversely, the strong deleterious effects of phthalates on germ cells were constantly present during the active phases of gonocyte development and thus share no relationship with the steroidogenic status. Moreover, all the effects of phthalates were unchanged in testes from mice deficient for estrogen (ERKO or ERβKO) or androgen (Tfm) receptors. In conclusion, our results demonstrate that phthalates impair mouse fetal germ cell number similarly to other mammalian species, but are neither estrogenic nor antiandrogenic molecules because their effects do not involve, directly or indirectly, ER or AR.

Upper Respiratory Tract Uptake of Naphthalene

Morris, J. B., Buckpitt, A. R. — Fri, 11 Sep 2009 17:40:58 PDT

Naphthalene is a nasal toxicant and carcinogen in the rat. Upper respiratory tract (URT) uptake of naphthalene was measured in the male and female F344 rat at exposure concentrations of 1, 4, 10, or 30 ppm at inspiratory flow rates of 150 or 300 ml/min. To assess the potential importance of nasal cytochrome P450 (CYP) metabolism, groups of rats were pretreated with the CYP inhibitor 5-phenyl-1-pentyne (PP) (100 mg/kg, ip). In vitro metabolism of naphthalene was similar in nasal tissues from both genders and was reduced by 80% by the inhibitor. URT uptake in female rats was concentration dependent with uptake efficiencies (flow 150 ml/min) of 56, 40, 34, and 28% being observed at inspired concentrations of 1, 4, 10, and 30 ppm, respectively. A similar effect was observed in male rats (flow 150 ml/min) with uptake efficiencies of 57, 49, 37, and 36% being observed. Uptake was more efficient in the male than female rat, likely due to their larger size (226 vs. 144 g). Uptake of naphthalene was significantly reduced by inhibitor pretreatment with the effect being greater at the lower inspired concentrations. Specifically, in pretreated female rats (150 ml/min), URT uptake averaged 25, 29, and 26% at inspired concentrations of 4, 10, and 30 ppm, respectively. Thus, the concentration dependence of uptake was virtually abolished by PP pretreatment. These results provide evidence that nasal CYP metabolism of naphthalene contributes to URT scrubbing of this vapor and is also involved in the concentration dependence of uptake that is observed.

Differential Pulmonary Retention of Diesel Exhaust Particles in Wistar Kyoto and Spontaneously Hypertensive Rats

Fri, 11 Sep 2009 17:40:58 PDT

Spontaneously hypertensive (SH) and normotensive Wistar Kyoto (WKY) rats have been used for understanding the mechanisms of variations in susceptibility to airborne pollutants. We examined the lung burden of diesel exhaust particles (DEP) following inhalation of diesel engine exhaust (DEE) in both strains. The kinetics of clearance was also examined after single intratracheal (IT) instillation of DEP. Lungs were analyzed for DEP elemental carbon (EC) after exposure to DEE (0, 500, or 2000 µg/m³ 4 h/day, 5 days/week x 4 weeks). SH rats had 16% less DEP-EC at 500 and 32% less at 2000 µg/m³ in the lungs, despite having 50% higher than the average minute volume. No strain-related differences were noted in number of alveolar macrophages or their average DEP load as evident from examining cells in bronchoalveolar lavage fluid (BALF). The kinetics of DEP clearance from lungs of male WKY and SH rats was studied following a single instillation at 0.0 or 8.33 mg/kg of DEP standard reference material (SRM 2975) from the National Institute of Standards Technology. SH rats cleared 60% DEP over 112 days while minimal clearance occurred from the lungs of WKY. The pattern of DEP-induced inflammatory response assessed by BALF analysis was similar in both strains, although the overall protein leak was slightly greater in SH rats. A time-dependent accumulation of DEP occurred in tracheal lymph nodes of both strains (SH > WKY). Thus, SH rats may clear DEP more efficiently from their lungs than normotensive WKY rats, with a small contribution of more effective lymphatic drainage.

Biomarkers of Drug-Induced Skeletal Muscle Injury in the Rat: Troponin I and Myoglobin

Fri, 11 Sep 2009 17:40:58 PDT

The purpose of this investigation was to determine the utility of fast-twitch skeletal muscle troponin I (fsTnI) and urinary myoglobin (uMB) as biomarkers of skeletal muscle injury in 8-week-old Sprague-Dawley rats. fsTnI and uMB were quantified by enzyme-linked immunosorbent assay and compared with standard clinical assays including creatine kinase, aldolase, aspartate aminotransferase, and histopathological assessments. Detectable levels of uMB were normalized to urinary creatinine to control for differences in renal function. Seven compounds, including those with toxic effects on skeletal muscle, cardiac muscle, or liver, were evaluated. fsTnI was typically nondetectable (< 5.9 ng/ml serum) in vehicle-treated female and male rats but increased in a dose-dependent manner to at least 300 ng/ml in cerivastatin-induced severe fast-twitch specific myotoxicity. Minimal myopathy induced by investigational compounds BMS-600149 and BMS-687453 increased serum fsTnI to about 30–50 ng/ml, suggesting a reasonable dynamic range for detecting mild to severe skeletal muscle toxicity. In direct contrast, fsTnI was only marginally increased relative to population control values in rats treated with triamcinolone acetonide, which produces muscle atrophy or the cardiotoxins isoproterenol and CoCl₂. uMB was typically nondetectable (< 1.6 ng/ml urine) in vehicle-treated female and male rats but increased to approximately 140, 300, and 30 ng/mg creatinine in rats treated with cerivastatin, BMS-687453, and triamcinolone acetonide, respectively. Cardiotoxicity also increased uMB in rats treated with isoproterenol and CoCl₂ with urine concentrations ranging from 20 to 30 ng/mg creatinine. Severe hepatotoxicity (coumarin) did not significantly affect serum fsTnI or uMB levels. Collectively, these data suggest that fsTnI is specific for skeletal muscle toxicity, whereas uMB is nonspecific, increasing with skeletal muscle and cardiac toxicity. Accordingly, the complement of fsTnI and uMB, in conjunction with standard clinical assays may comprise a useful diagnostic panel for assessing drug-induced myopathy in rats.

Inhibition of p38-MAPK Potentiates Cisplatin-Induced Apoptosis via GSH Depletion and Increases Intracellular Drug Accumulation in Growth-Arrested Kidney Tubular Epithelial Cells

Fri, 11 Sep 2009 17:40:58 PDT

We were interested in analyzing the regulation by mitogen-activated protein kinases (MAPKs) of cisplatin-provoked toxicity in epithelial renal tubule cell lines, when assayed under culture conditions (cell confluence plus serum deprivation), which mimic the characteristics of a nonproliferating epithelium. Under these restrictive growth conditions, cisplatin induced apoptosis with lower efficacy than in exponentially growing cells, and decreased p38-MAPK phosphorylation in NRK-52E and other (LLC-PK1, MDCK, HK2) cell lines. Moreover, cisplatin-provoked apoptosis was potentiated by cotreatment with p38-MAPK–specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, whereas c-Jun NH₂-terminal kinase or extracellular signal-regulated kinase/MAPK and ERK Kinase inhibitors were ineffective. By contrast, when applied to exponentially growing cells, cisplatin stimulated p38-MAPK phosphorylation and apoptosis, was attenuated by kinase inhibitors. Treatment of confluent/serum-deprived cells with cisplatin caused mitochondrial transmembrane potential disruption and activated the mitochondrial apoptotic pathway, as indicated by the decrease in Bcl-X_L expression, increase in Bax expression and cytochrome c release, and these effects were potentiated by cotreatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular reduced glutathione (GSH) content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Cotreatment with the GSH-depleting agent D,L-buthionine-R,S-sulfoximine also potentiated cisplatin-provoked apoptosis. In summary, p38-MAPK inhibition potentiates cisplatin-provoked apoptosis in growth-arrested epithelial renal tubule cells, a result that may be explained at least in part by GSH depletion and drug transport alteration.

Comparative Functional Genomic Analysis Identifies Distinct and Overlapping Sets of Genes Required for Resistance to Monomethylarsonous Acid (MMAIII) and Arsenite (AsIII) in Yeast

Fri, 11 Sep 2009 17:40:58 PDT

Arsenic is a human toxin and carcinogen commonly found as a contaminant in drinking water. Arsenite (As^III) is the most toxic inorganic form, but recent evidence indicates that the metabolite monomethylarsonous acid (MMA^III) is even more toxic. We have used a chemical genomics approach to identify the genes that modulate the cellular toxicity of MMA^III and As^III in the yeast Saccharomyces cerevisiae. Functional profiling using homozygous deletion mutants provided evidence of the requirement of highly conserved biological processes in the response against both arsenicals including tubulin folding, DNA double-strand break repair, and chromatin modification. At the equitoxic doses of 150µM MMA^III and 300µM As^III, genes related to glutathione metabolism were essential only for resistance to the former, suggesting a higher potency of MMA^III to disrupt glutathione metabolism than As^III. Treatments with MMA^III induced a significant increase in glutathione levels in the wild-type strain, which correlated to the requirement of genes from the sulfur and methionine metabolic pathways and was consistent with the induction of oxidative stress. Based on the relative sensitivity of deletion strains deficient in GSH metabolism and tubulin folding processes, oxidative stress appeared to be the primary mechanism of MMA^III toxicity whereas secondary to tubulin disruption in the case of As^III. Many of the identified yeast genes have orthologs in humans that could potentially modulate arsenic toxicity in a similar manner as their yeast counterparts.

Actin-Mediated Endocytosis Limits Intracellular Cr Accumulation and Cr Toxicity during Chromate Stress

Holland, S. L., Avery, S. V. — Fri, 11 Sep 2009 17:40:58 PDT

Chromate toxicity is well documented, but the underlying toxic mechanism(s) has yet to be fully elucidated. Following a Cr toxicity screen against > 6000 heterozygous yeast mutants, here we show that Cr resistance requires normal function of the cortical actin cytoskeleton. Furthermore, Cr-stressed yeast cells exhibited an increased number of actin patches, the sites of endocytosis. This was coincident with a marked stimulation of endocytosis following Cr exposure. Genetic dissection of actin nucleation from endocytosis revealed that endocytosis, specifically, was required for Cr resistance. A series of further endocytosis mutants (sac6, chc1, end3) exhibited elevated Cr sensitivity. These mutants also showed markedly elevated cellular Cr accumulation, explaining their sensitivities. In wild-type cells, an initial endocytosis-independent phase of Cr uptake was followed by an endocytosis-dependent decline in Cr accumulation. The results indicate that actin-mediated endocytosis is required to limit Cr accumulation and toxicity. It is proposed that this involves ubiquitin-dependent endocytic inactivation of a plasma membrane Cr transporter(s). We showed that such an action was not dependent on the transporters that have been characterized to date, the sulfate (and chromate) permeases Sul1p and Sul2p.

Toxicological Sciences - recent issues

TABLE OF CONTENTS

COVER

EDITORIAL BOARD

SUBSCRIPTIONS

Long-term Inhalation Toxicity Studies with Multiwalled Carbon Nanotubes: Closing the Gaps or Initiating the Debate?

State of Academic Knowledge on Toxicity and Biological Fate of Quantum Dots

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

Modulation of Aflatoxin B1-Mediated Genotoxicity in Primary Cultures of Human Hepatocytes by Diindolylmethane, Curcumin, and Xanthohumols

Use of Short-term Transcriptional Profiles to Assess the Long-term Cancer-Related Safety of Environmental and Industrial Chemicals

S-Adenosylhomocysteine Promotes the Invasion of C6 Glioma Cells via Increased Secretion of Matrix Metalloproteinase-2 in Murine Microglial BV2 Cells

Mal-Development of the Penis and Loss of Fertility in Male Rats Treated Neonatally with Female Contraceptive 17{alpha}-Ethinyl Estradiol: A Dose-Response Study and a Comparative Study with a Known Estrogenic Teratogen Diethylstilbestrol

Dynamic Nature of Alterations in the Endocrine System of Fathead Minnows Exposed to the Fungicide Prochloraz

Gene and Protein Responses of Human Monocytes to Extracellular Cysteine Redox Potential

Establishment of an Immunoglobulin M Antibody-Forming Cell Response Model for Characterizing Immunotoxicity in Primary Human B Cells

Discrimination for Genotoxic and Nongenotoxic Carcinogens by Gene Expression Profiling in Primary Mouse Hepatocytes Improves with Exposure Time

Weighted Feature Significance: A Simple, Interpretable Model of Compound Toxicity Based on the Statistical Enrichment of Structural Features

Age-Dependent Susceptibility to Manganese-Induced Neurological Dysfunction

Developmental Exposure to Manganese Increases Adult Susceptibility to Inflammatory Activation of Glia and Neuronal Protein Nitration

Probucol Increases Glutathione Peroxidase-1 Activity and Displays Long-Lasting Protection against Methylmercury Toxicity in Cerebellar Granule Cells

Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism

Metallothionein-III Provides Neuronal Protection through Activation of Nuclear Factor-{kappa}B via the TrkA/Phosphatidylinositol-3 kinase/Akt Signaling Pathway

Early-Life Exposure to Dimethoate-Induced Reproductive Toxicity: Evaluation of Effects on Pituitary-Testicular Axis of Mice

Reproductive and Sphingolipid Metabolic Effects of Fumonisin B1 and its Alkaline Hydrolysis Product in LM/Bc Mice: Hydrolyzed Fumonisin B1 Did Not Cause Neural Tube Defects

Inhalation Toxicity of Multiwall Carbon Nanotubes in Rats Exposed for 3 Months

Acute Morphological and Toxicological Effects in a Human Bronchial Coculture Model after Sulfur Mustard Exposure

Estimates of Cancer Potency of 2,3,7,8-Tetrachlorodibenzo(p)dioxin Using Linear and Nonlinear Dose-Response Modeling and Toxicokinetics

Toxicogenomics Applied to Cultures of Human Hepatocytes Enabled an Identification of Novel Petasites hybridus Extracts for the Treatment of Migraine with Improved Hepatobiliary Safety

High-Mobility Group Box-1 Protein and Keratin-18, Circulating Serum Proteins Informative of Acetaminophen-Induced Necrosis and Apoptosis In Vivo

A Single Exposure to Particulate or Gaseous Air Pollution Increases the Risk of Aconitine-Induced Cardiac Arrhythmia in Hypertensive Rats

CONTENTS

COVER

Editorial Board

SUBSCRIPTIONS

Hypoxia Response: A Model Toxicity Pathway for High-Throughput Screening

Nitrative and Oxidative Stress in Toxicology and Disease

Endless Possibilities: Stem Cells and the Vision for Toxicology Testing in the 21st Century

Lactational Transfer of Manganese in Rats: Predicting Manganese Tissue Concentration in the Dam and Pups from Inhalation Exposure with a Pharmacokinetic Model

Evaluating Placental Transfer and Tissue Concentrations of Manganese in the Pregnant Rat and Fetuses after Inhalation Exposures with a PBPK Model

3-Methylindole is Mutagenic and a Possible Pulmonary Carcinogen

An Evaluation of Estrogenic Activity of Parabens Using Uterine Calbindin-D9k Gene in an Immature Rat Model

Chlorotriazine Herbicides and Metabolites Activate an ACTH-dependent Release of Corticosterone in Male Wistar Rats

Characterization of the Hypothalamic-Pituitary-Adrenal Axis Response to Atrazine and Metabolites in the Female Rat

Comparative Study on Susceptibility to 1-Bromopropane in Three Mice Strains

Urban Particulate Matter Causes ER Stress and the Unfolded Protein Response in Human Lung Cells

Simultaneous In Vivo Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS-stimulated Primary IgM Response

Gestational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Disrupts B-Cell Lymphopoiesis and Exacerbates Autoimmune Disease in 24-Week-Old SNF1 Mice

Mast Cells Mediate the Immune Suppression Induced by Dermal Exposure to JP-8 Jet Fuel

Identification of Chemical Compounds that Induce HIF-1{alpha} Activity

Investigation of Peptide Reactivity of Pro-hapten Skin Sensitizers Using a Peroxidase-Peroxide Oxidation System

Effects of Methyl Mercury in Combination with Polychlorinated Biphenyls and Brominated Flame Retardants on the Uptake of Glutamate in Rat Brain Synaptosomes: A Mathematical Approach for the Study of Mixtures

Long-term Cognitive Impairments in Adult Rats Treated Neonatally with {beta}-N-Methylamino-L-Alanine

Investigations of the Use of Bioavailability Data to Adjust Occupational Exposure Limits for Active Pharmaceutical Ingredients

N-acetyltransferase 2 Genotype Modification of Active Cigarette Smoking on Breast Cancer Risk among Hispanic and Non-Hispanic White Women

Automated Dose-Response Analysis of the Relative Hepatic Gene Expression Potency of TCDF in C57BL/6 Mice

Comparative Analysis of AhR-Mediated TCDD-Elicited Gene Expression in Human Liver Adult Stem Cells

Dioxin-Dependent and Dioxin-Independent Gene Batteries: Comparison of Liver and Kidney in AHR-Null Mice

Divergent Transcriptomic Responses to Aryl Hydrocarbon Receptor Agonists between Rat and Human Primary Hepatocytes

Editorial Board

SUBSCRIPTIONS

Cross-talk between Transcription Factors AhR and Nrf2: Lessons for Cancer Chemoprevention from Dioxin

Cellular Stress Response Pathway System as a Sentinel Ensemble in Toxicological Screening

Biologically Relevant Exposure Science for 21st Century Toxicity Testing

Are In Vitro Tests Suitable for Regulatory Use?

Introducing the "TCDD-Inducible AhR-Nrf2 Gene Battery"

Effect of Dietary Selenium and Cigarette Smoke on Pulmonary Cell Proliferation in Mice

Dioxin Increases the Interaction Between Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha at Human Promoters

Roles of the Genetic Polymorphisms of Alcohol-Metabolizing Enzymes on the Immunology in High-Risk Drinkers

Induction of Suppressors of Cytokine Signaling by the Trichothecene Deoxynivalenol in the Mouse

Trovafloxacin Enhances TNF-Induced Inflammatory Stress and Cell Death Signaling and Reduces TNF Clearance in a Murine Model of Idiosyncratic Hepatotoxicity

Halothane-Induced Liver Injury is Mediated by Interleukin-17 in Mice

Effects of 18 Perfluoroalkyl Compounds on mRNA Expression in Chicken Embryo Hepatocyte Cultures

MRI Characterization of Structural Mouse Brain Changes in Response to Chronic Exposure to the Glufosinate Ammonium Herbicide

Iron-Induced Oxidative Injury Differentially Regulates PI3K/Akt/GSK3{beta} Pathway in Synaptic Endings from Adult and Aged Rats

Caenorhabditis elegans Metallothioneins Protect against Toxicity Induced by Depleted Uranium

A Minimally Invasive, Translational Biomarker of Ketamine-Induced Neuronal Death in Rats: microPET Imaging Using 18F-Annexin V

Toluene-Induced Hearing Loss in the Guinea Pig

Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism

Upper Respiratory Tract Uptake of Naphthalene

Differential Pulmonary Retention of Diesel Exhaust Particles in Wistar Kyoto and Spontaneously Hypertensive Rats