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1.  Molecular Mechanisms of Hepatocellular Apoptosis Induced by Trovafloxacin-Tumor Necrosis Factor-alpha Interaction 
Toxicological Sciences  2013;137(1):91-101.
Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.
doi:10.1093/toxsci/kft226
PMCID: PMC3871929  PMID: 24097668
idiosyncratic drug-induced liver injury; hepatotoxicity; caspase; JNK; trovafloxacin.
2.  Cumulative Risk: Toxicity and Interactions of Physical and Chemical Stressors 
Toxicological Sciences  2013;137(1):3-11.
Recent efforts to update cumulative risk assessment procedures to incorporate nonchemical stressors ranging from physical to psychosocial reflect increased interest in consideration of the totality of variables affecting human health and the growing desire to develop community-based risk assessment methods. A key roadblock is the uncertainty as to how nonchemical stressors behave in relationship to chemical stressors. Physical stressors offer a reasonable starting place for measuring the effects of nonchemical stressors and their modulation of chemical effects (and vice versa), as they clearly differ from chemical stressors; and “doses” of many physical stressors are more easily quantifiable than those of psychosocial stressors. There is a commonly held belief that virtually nothing is known about the impact of nonchemical stressors on chemically mediated toxicity or the joint impact of coexposure to chemical and nonchemical stressors. Although this is generally true, there are several instances where a substantial body of evidence exists. A workshop titled “Cumulative Risk: Toxicity and Interactions of Physical and Chemical Stressors” held at the 2013 Society of Toxicology Annual Meeting provided a forum for discussion of research addressing the toxicity of physical stressors and what is known about their interactions with chemical stressors, both in terms of exposure and effects. Physical stressors including sunlight, heat, radiation, infectious disease, and noise were discussed in reference to identifying pathways of interaction with chemical stressors, data gaps, and suggestions for future incorporation into cumulative risk assessments.
doi:10.1093/toxsci/kft228
PMCID: PMC3871930  PMID: 24154487
cumulative risk; nonchemical stressors; joint action; radiation; infectious disease; sunlight; temperature; noise.
3.  The Adaptive Endoplasmic Reticulum Stress Response to Lipotoxicity in Progressive Human Nonalcoholic Fatty Liver Disease 
Toxicological Sciences  2013;137(1):26-35.
Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7%–14% of the U.S. population through a second “hit” in the form of increased oxidative stress and inflammation. Endoplasmic reticulum (ER) stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress–associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty), and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0 ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis, and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress–associated lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress–associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor X-box binding protein 1 spliced (XBP-1s) were significantly elevated among NASH samples, whereas other downstream ER stress proteins including CHOP, ATF4, and phosphorylated JNK and eIF2α were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD.
doi:10.1093/toxsci/kft230
PMCID: PMC3871931  PMID: 24097666
nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; lipotoxicity; endoplasmic reticulum stress response mechanisms.
4.  Multidimensional In Vivo Hazard Assessment Using Zebrafish 
Toxicological Sciences  2013;137(1):212-233.
Editor’s Highlight: The Tanguay group uses the embryonic zebrafish model to demonstrate the utility of high throughput screening for toxicology studies. The group evaluated the 1060 US EPA ToxCast Phase 1 and 2 compounds on 18 distinct outcomes. With four doses for each compound the group generated a dizzying number of data points highlighting the importance of bioinformatics analysis in these types of studies. The study shows how it is now possible to screen many of the tens of thousands of untested chemicals using a whole animal model in which one can literally see developmental malformations. —Gary W. Miller
There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.
doi:10.1093/toxsci/kft235
PMCID: PMC3871932  PMID: 24136191
developmental; high-throughput screening; Tox21; ToxCast.
5.  Aberrant Methylation of Hypermethylated-in-Cancer-1 and Exocyclic DNA Adducts in Tobacco Smokers 
Toxicological Sciences  2013;137(1):47-54.
Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M1dG (a malondialdehyde adduct), measured by 32P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p ≤ .05). A dose response was observed, with the highest HIC1 methylation levels in smokers of ≥ 10 cigarettes/day relative to nonsmokers and intermediate values in smokers of 1–9 cigarettes/day (p for trend ≤ .001). No additional relationships were observed. We also evaluated correlations between M1dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M1dG (covariate-adjusted regression coefficient (β) = .224 ± .101 [SE], p ≤ .05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M1dG in connecting smoking to this latter hypermethylation.
doi:10.1093/toxsci/kft241
PMCID: PMC3871933  PMID: 24154486
tobacco smoking; overall and site specific methylation; HIC1; oxidative DNA damage; M1dG.
6.  The Nature of Nurture: Refining the Definition of the Exposome 
Toxicological Sciences  2013;137(1):1-2.
doi:10.1093/toxsci/kft251
PMCID: PMC3871934  PMID: 24213143
exposome; genome; environment.
7.  Nucleotide Specificity of DNA Binding of the Aryl Hydrocarbon Receptor:ARNT Complex Is Unaffected by Ligand Structure 
Toxicological Sciences  2013;137(1):102-113.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and a wide variety of structurally diverse ligands through its ability to translocate into the nucleus and bind to a specific DNA recognition site (the dioxin-responsive element [DRE]) adjacent to responsive genes. Although the sequence of the DRE is well defined, several reports suggested that the nucleotide specificity of AhR DNA binding may vary depending on the structure of its bound ligand. Given the potential toxicological significance of this hypothesis, an unbiased DNA-selection-and-PCR-amplification approach was utilized to directly determine whether binding and activation of the AhR by structurally diverse agonists alter its nucleotide specificity of DNA binding. Guinea pig hepatic cytosolic AhR activated in vitro by equipotent concentrations of TCDD, 3-methylcholanthrene, β-naphthoflavone, indirubin, L-kynurenine, or YH439 was incubated with a pool of DNA oligonucleotides containing a 15-base pair variable region consisting of all possible nucleotides. The AhR-bound oligonucleotides isolated by immunoprecipitation were PCR amplified and used in subsequent rounds of selection. Sequence analysis of a total of 196 isolated oligonucleotides revealed that each ligand-activated AhR:ARNT complex only bound to DRE-containing DNA oligonucleotides; no non-DRE-containing DNA oligonucleotides were identified. These results demonstrate that the binding and activation of the AhR by structurally diverse agonists do not appear to alter its nucleotide specificity of DNA binding and suggest that stimulation of gene expression mediated by direct DNA binding of ligand-activated AhR:ARNT complexes is DRE dependent.
doi:10.1093/toxsci/kft234
PMCID: PMC3924043  PMID: 24136190
Ah Receptor; TCDD; DRE; dioxin.
8.  Early Life Manganese Exposure Upregulates Tumor-Associated Genes in the Hypothalamus of Female Rats: Relationship to Manganese-Induced Precocious Puberty 
Toxicological Sciences  2013;136(2):373-381.
Prepubertal exposure to low, but elevated levels of manganese (Mn) can induce increased secretions of puberty-related hormones resulting in precocious pubertal development in female rats. These events are due to an action of the element within the hypothalamus to induce the secretion of gonadotropin-releasing hormone (GnRH). Because of these prepubertal effects of Mn and because precocious puberty is a serious neuroendocrine disorder, we have assessed whether early life exposure to this environmental element is capable of precociously upregulating the expression of a select group of genes previously associated with tumor growth or suppression, and that have more recently been shown to increase at the normal time of puberty. Female rat pups received a daily dose of either 10mg/kg manganese(II) chloride or an equal volume of saline by gastric gavage from postnatal day 12 through day 22 or 29. At this time, blood was collected for estradiol analysis and hypothalamic brain tissue frozen on dry ice until assessed for gene expressions. Rats exposed to the elevated levels of Mn showed a precocious increase in GnRH gene expression in the preoptic area and rostral hypothalamus on day 29, an action associated with precociously increased expressions of specific tumor-associated, puberty-related genes. These results demonstrate for the first time that prepubertal Mn exposure is capable of activating specific upstream genes regulating hypothalamic GnRH and suggest that these actions are involved in the mechanism by which this element can induce precocious puberty.
doi:10.1093/toxsci/kft195
PMCID: PMC3858189  PMID: 23997110
manganese; puberty; KiSS-1; Ras homologue enriched in brain; mammalian target of rapamycin.
9.  Sodium Methyldithiocarbamate Exerts Broad Inhibition of Cellular Signaling and Expression of Effector Molecules of Inflammation 
Toxicological Sciences  2013;136(2):430-442.
Sodium methyldithiocarbamate (SMD) is one of the most abundantly used conventional pesticides in the United States. At dosages relevant to occupational exposure, it causes major effects on the immune system in mice, including a decreased resistance to sepsis. This lab has identified some of the mechanisms of action of this compound and some of the immunological parameters affected, but the global effects have not previously been assessed. The purpose of the present study was to conduct transcriptomic analysis of the effects of SMD on lipopolysaccharide-induced expression of mediators important in innate immunity and inflammation. The results revealed broad effects on expression of transcription factors in both branches of Toll-like receptor 4 (TLR4) signaling (MyD88 and TRIF). However, TLR3 and interferon signaling pathways were decreased to a greater extent, and assessment of the effects of SMD on polyinosinic polycytidylic acid–induced cytokine and chemokine production revealed that these responses mediated by TLR3 were indeed sensitive to the effects of SMD, with inhibition occurring at lower dosages than required to inhibit responses to other immunological stimuli tested in our previous studies. In the downstream signaling pathways of these TLRs, functional analysis also revealed that NF-κB activation was inhibited by SMD, as indicated by gene expression analysis and a reporter construct in mice. A previously unreported effect on luteinizing hormone and follicle-stimulating hormone pathways was also observed.
doi:10.1093/toxsci/kft196
PMCID: PMC3858190  PMID: 24056979
pesticides; signal transduction; transcription factors; chemokines; cytokine; signaling; microarray.
10.  Structure-Activity Relationship of Selected Meta- and Para-Hydroxylated Non–Dioxin Like Polychlorinated Biphenyls: From Single RyR1 Channels to Muscle Dysfunction 
Toxicological Sciences  2013;136(2):500-513.
Non–dioxin like polychlorinated biphenyls (NDL-PCBs) are legacy environmental contaminants with contemporary unintentional sources. NDL-PCBs interact with ryanodine receptors (RyRs), Ca2+ channels of sarcoplasmic/endoplasmic reticulum (SR/ER) that regulate excitation-contraction coupling (ECC) and Ca2+-dependent cell signaling in muscle. Activities of 4 chiral congeners PCB91, 95, 132, and 149 and their respective 4- and 5-hydroxy (-OH) derivatives toward rabbit skeletal muscle ryanodine receptor (RyR1) are investigated using [3H]ryanodine binding and SR Ca2+ flux analyses. Although 5-OH metabolites have comparable activity to their respective parent in both assays, 4-OH derivatives are unable to trigger Ca2+ release from SR microsomes in the presence of Ca2+-ATPase activity. PCB95 and derivatives are investigated using single channel voltage-clamp and primary murine embryonic muscle cells (myotubes). Like PCB95, 5-OH-PCB95 quickly and persistently increases channel open probability (p o > .9) by stabilizing the full-open channel state, whereas 4-OH-PCB95 transiently enhances p o. Ca2+ imaging of myotubes loaded with Fluo-4 show that acute exposure to PCB95 (5µM) potentiates ECC and caffeine responses and partially depletes SR Ca2+ stores. Exposure to 5-OH-PCB95 (5 µM) increases cytoplasmic Ca2+, leading to rapid ECC failure in 50% of myotubes with the remainder retaining negligible responses. 4-OH-PCB95 neither increases baseline Ca2+ nor causes ECC failure but depresses ECC and caffeine responses by 50%. With longer (3h) exposure to 300nM PCB95, 5-OH-PCB95, or 4-OH-PCB95 decreases the number of ECC responsive myotubes by 22%, 81%, and 51% compared with control by depleting SR Ca2+ and/or uncoupling ECC. NDL-PCBs and their 5-OH and 4-OH metabolites differentially influence RyR1 channel activity and ECC in embryonic skeletal muscle.
doi:10.1093/toxsci/kft202
PMCID: PMC3858193  PMID: 24014653
calcium signaling; ryanodine receptor; muscle dysfunction; polychlorinated biphenyls; hydroxylated metabolites.
11.  SOT Symposium Highlight: Translatable Indicators of Testicular Toxicity: Inhibin B, MicroRNAs, and Sperm Signatures 
Toxicological Sciences  2013;136(2):265-273.
Testicular toxicity is an important safety endpoint in drug development and risk assessment, but reliable and translatable biomarkers for predicting injury have eluded researchers. However, this area shows great potential for improvement, with several avenues currently being pursued. This was the topic of a symposium session during the 2013 Society of Toxicology Annual Meeting in San Antonio, TX, entitled “Translatable Indicators of Testicular Toxicity: Inhibin B, MicroRNAs, and Sperm Signatures.” This symposium brought together stakeholders from academia, government, and industry to present the limitations and drawbacks of currently used indicators of injury and discussed the ongoing efforts in developing more predictive biomarkers of injury. The presentations highlighted the early challenges of using circulating inhibin B and microRNA levels, and sperm messenger RNA transcript abundance and DNA methylation profiles, as novel biomarkers of testicular toxicity.
doi:10.1093/toxsci/kft207
PMCID: PMC3858194  PMID: 24052563
biomarkers; testicular toxicity; inhibin B; microRNA; sperm.
12.  A Single Gestational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Disrupts the Adult Uterine Response to Estradiol in Mice 
Toxicological Sciences  2013;136(2):514-526.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) given as a cotreatment with estrogen exhibits antiestrogenic properties on the rodent adult uterus, but less is understood regarding hormonal responsiveness of the adult uterus from animals having been exposed to TCDD during critical periods of development. We characterized the inhibitory effects of TCDD (T) exposure at gestational day 15 (GD15), 4 weeks, and 9 weeks of age (TTT) on the adult uterus following hormone treatment. TTT-exposed mice in response to hormone treatment exhibited a blunted weight increase, had fewer uterine glands, displayed morphological anomalies, and had marked decreases in the hormonal regulation of genes involved in fluid transport (Aqp3 and Aqp5), cytoarchitectural (Dsc2 and Sprr2A), and immune (Lcn2 and Ltf) regulation. To determine if the 9-week exposure was responsible for the blunted uterine response, due to the 7- to 11-day half-life of TCDD in mice, a second set of experiments was performed to examine exposure to TCDD given at GD15, GD15 only (cross-fostered at birth), only during lactation (cross-fostered at birth), or at GD15 and 4 weeks of age. Our studies demonstrate that a single developmental TCDD exposure at GD15 is sufficient to elicit a blunted adult uterine response to estradiol and is due in part to fewer gland numbers and the reduced expression of forkhead box A2 (FoxA2), a gene involved in gland development. Together, these results provide insight regarding the critical nature of in utero exposure and the potential impact on ensuing uterine biology and reproductive health later in life.
doi:10.1093/toxsci/kft208
PMCID: PMC3858195  PMID: 24052564
TCDD; uterus; developmental exposure.
13.  Uptake, p53 Pathway Activation, and Cytotoxic Responses for Co(II) and Ni(II) in Human Lung Cells: Implications for Carcinogenicity 
Toxicological Sciences  2013;136(2):467-477.
Cobalt(II) and nickel(II) ions display similar chemical properties and act as hypoxia mimics in cells. However, only soluble Co(II) but not soluble Ni(II) is carcinogenic by inhalation. To explore potential reasons for these differences, we examined responses of human lung cells to both metals. We found that Co(II) showed almost 8 times higher accumulation than Ni(II) in H460 cells but caused a less efficient activation of the transcriptional factor p53 as measured by its accumulation, Ser15 phosphorylation, and target gene expression. Unlike Ni(II), Co(II) was ineffective in downregulating the p53 inhibitor MDM4 (HDMX). Co(II)-treated cells continued DNA replication at internal doses that caused massive apoptosis by Ni(II). Apoptosis and the overall cell death by Co(II) were delayed and weaker than by Ni(II). Inhibition of caspases but not programmed necrosis pathways suppressed Co(II)-induced cell death. Knockdown of p53 produced 50%–60% decreases in activation of caspases 3/7 and expression of 2 most highly upregulated proapoptotic genes PUMA and NOXA by Co(II). Overall, p53-mediated apoptosis accounted for 55% cell death by Co(II), p53-independent apoptosis for 20%, and p53/caspase-independent mechanisms for 25%. Similar to H460, normal human lung fibroblasts and primary human bronchial epithelial cells had several times higher accumulation of Co(II) than Ni(II) and showed a delayed and weaker caspase activation by Co(II). Thus, carcinogenicity of soluble Co(II) could be related to high survival of metal-loaded cells, which permits accumulation of genetic and epigenetic abnormalities. High cytotoxicity of soluble Ni(II) causes early elimination of damaged cells and is expected to be cancer suppressive.
doi:10.1093/toxsci/kft214
PMCID: PMC3858198  PMID: 24068677
cobalt; nickel; toxicity; cancer; TP53; apoptosis.
14.  Stimulation-Evoked Dopamine Release in the Nucleus Accumbens Following Cocaine Administration in Rats Perinatally Exposed to Polychlorinated Biphenyls 
Toxicological Sciences  2013;136(1):144-153.
Exposure to polychlorinated biphenyls (PCBs) alters brain dopamine (DA) concentrations and DA receptor/transporter function, suggesting the reinforcing properties of drugs of abuse acting on the DA system may be affected by PCB exposure. Female Long-Evans rats were orally exposed to 0, 3, or 6mg/kg/day PCBs from 4 weeks prior to breeding until litters were weaned on postnatal day 21. In vivo fixed potential amperometry (FPA) was used in adult anesthetized offspring to determine whether perinatal PCB exposure altered (1) presynaptic DA autoreceptor (DAR) sensitivity, (2) electrically evoked nucleus accumbens (NAc) DA efflux following administration of cocaine, and (3) the rate of depletion of presynaptic DA stores. One adult male and female littermate were tested using FPA following a single injection of cocaine (20mg/kg ip), whereas a second adult male and female littermate were tested following the last of seven daily cocaine injections of the same dose. The carbon fiber recording microelectrode was positioned in the NAc core, and DA oxidation currents (i.e., DA release) evoked by brief stimulation of the medial forebrain bundle (MFB) were quantified before and after administration of cocaine. PCB-exposed rats exhibited enhanced stimulation-evoked DA release (relative to baseline) following a single injection of cocaine. Although nonexposed controls exhibited typical DA sensitization following repeated cocaine administration, this effect was attenuated in PCB-exposed rats. In addition, DAR sensitivity was higher (males only), and the rate of depletion of presynaptic DA stores was greater in PCB-exposed animals relative to nonexposed controls. These results indicate that perinatal PCB exposure can modify DA synaptic transmission in the NAc in a manner previously shown to alter the reinforcing properties of cocaine.
doi:10.1093/toxsci/kft171
PMCID: PMC3829567  PMID: 23912914
neurotoxicology; behavioral sensitization.
15.  The Nrf2 Activator, tBHQ, Differentially Affects Early Events Following Stimulation of Jurkat Cells 
Toxicological Sciences  2013;136(1):63-71.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4+ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4+ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca2+ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.
doi:10.1093/toxsci/kft172
PMCID: PMC3829568  PMID: 23945499
Nrf2; IL-2; calcium; NFĸB; tBHQ; T cell.
16.  Pretreatment With TCDD Exacerbates Liver Injury From Concanavalin A: Critical Role for NK Cells 
Toxicological Sciences  2013;136(1):72-85.
For many liver diseases, including viral and autoimmune hepatitis, immune cells play an important role in the development and progression of liver injury. Concanavalin A (Con A) administration to rodents has been used as a model of immune-mediated liver injury resembling human autoimmune hepatitis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to alter the development of immune-mediated diseases. Mice pretreated with TCDD developed exacerbated liver injury in response to administration of a mild dose (6mg/kg) of Con A. In the present study, we tested the hypothesis that TCDD pretreatment exacerbates Con A-induced liver injury by enhancing the activation and recruitment of accessory cell types including neutrophils, macrophages, and natural killer (NK) cells. Mice were treated with 0, 0.3, 3, or 30 μg/kg TCDD and 4 days later with Con A or saline. TCDD pretreatment with doses of 3 and 30 μg/kg significantly increased liver injury from Con A administration. The plasma concentrations of neutrophil chemokines were significantly increased in TCDD-pretreated mice after Con A administration. NKT cell-deficient (CD1d KO) mice were used to examine whether NKT cells were required for TCDD/Con A-induced liver injury. CD1d KO mice were completely protected from liver injury induced by treatment with Con A alone, whereas the injury from TCDD/Con A treatment was reduced but not eliminated. However, T-cell deficient (RAG1 KO) mice were protected from liver injury induced by Con A irrespective of pretreatment with TCDD. TCDD/Con A treatment increased the percentage of NK cells expressing the activation marker CD69. Depletion of NK cells prior to treatment resulted in significant reductions in plasma interferon-γ and liver injury from TCDD/Con A treatment. In summary, exposure to TCDD exacerbated the immune-mediated liver injury induced by Con A, and our findings suggest that NK cells play a critical role in this response.
doi:10.1093/toxsci/kft174
PMCID: PMC3829569  PMID: 23970800
dioxin; autoimmune; inflammation; chemokines; liver.
17.  Roles of the Hemostatic System and Neutrophils in Liver Injury From Co-exposure to Amiodarone and Lipopolysaccharide 
Toxicological Sciences  2013;136(1):51-62.
It has been demonstrated that co-treatment of rats with amiodarone (AMD) and bacterial lipopolysaccharide (LPS) produces idiosyncrasy-like liver injury. In this study, the hypothesis that the hemostatic system and neutrophils contribute to AMD/LPS-induced liver injury was explored. Rats were treated with AMD (400mg/kg, ip) or vehicle and 16h later with LPS (1.6×106 endotoxin units/kg, iv) or saline (Sal). AMD did not affect the hemostatic system by itself but significantly potentiated LPS-induced coagulation activation and fibrinolysis impairment. Increased hepatic fibrin deposition and subsequent hypoxia were observed only in AMD/LPS-treated animals, starting before the onset of liver injury. Administration of anticoagulant heparin abolished AMD/LPS-induced hepatic fibrin deposition and reduced AMD/LPS-induced liver damage. Polymorphonuclear neutrophils (PMNs) accumulated in liver after treatment with LPS or AMD/LPS, but PMN activation was only observed in AMD/LPS-treated rats. Rabbit anti-rat PMN serum, which reduced accumulation of PMNs in liver, prevented PMN activation and attenuated AMD/LPS-induced liver injury in rats. PMN depletion did not affect hepatic fibrin deposition. Anticoagulation prevented PMN activation without affecting PMN accumulation. In summary, both the hemostatic system alteration and PMN activation contributed to AMD/LPS-induced liver injury in rats, in which fibrin deposition was critical for the activation of PMNs.
doi:10.1093/toxsci/kft170
PMCID: PMC3829566  PMID: 23912913
amiodarone; idiosyncratic drug-induced liver injury; hemostatic system; neutrophils; hypochlorous acid.
18.  SLCO1B1 Variants and Urine Arsenic Metabolites in the Strong Heart Family Study 
Toxicological Sciences  2013;136(1):19-25.
Arsenic species patterns in urine are associated with risk for cancer and cardiovascular diseases. The organic anion transporter coded by the gene SLCO1B1 may transport arsenic species, but its association with arsenic metabolites in human urine has not yet been studied. The objective of this study is to evaluate associations of urine arsenic metabolites with variants in the candidate gene SLCO1B1 in adults from the Strong Heart Family Study. We estimated associations between % arsenic species biomarker traits and 5 single-nucleotide polymorphisms (SNPs) in the SLCO1B1 gene in 157 participants, assuming additive genetics. Linear regression models for each SNP accounted for kinships and were adjusted for sex, body mass index, and study center. The minor allele of rs1564370 was associated with lower %MMA (p = .0003) and higher %DMA (p = .0002), accounting for 8% of the variance for %MMA and 9% for %DMA. The rs1564370 minor allele homozygote frequency was 17% and the heterozygote frequency was 43%. The minor allele of rs2291075 was associated with lower %MMA (p = .0006) and higher %DMA (p = .0014), accounting for 7% of the variance for %MMA and 5% for %DMA. The frequency of rs2291075 minor allele homozygotes was 1% and of heterozygotes was 15%. Common variants in SLCO1B1 were associated with differences in arsenic metabolites in a preliminary candidate gene study. Replication of this finding in other populations and analyses with respect to disease outcomes are needed to determine whether this novel candidate gene is important for arsenic-associated disease risks.
doi:10.1093/toxsci/kft181
PMCID: PMC3829571  PMID: 23970802
American Indians; arsenic metabolism; arsenic species; SLCO1B1; OATPC; Strong Heart Study.
19.  A New Player in Environmentally Induced Oxidative Stress: Polychlorinated Biphenyl Congener, 3,3′-Dichlorobiphenyl (PCB11) 
Toxicological Sciences  2013;136(1):39-50.
Recent analysis of air samples from Chicago and Lake Michigan areas observed a ubiquitous airborne polychlorinated biphenyl (PCB) congener, 3,3′-dichlorobiphenyl (PCB11). Our analysis of serum samples also revealed the existence of hydroxylated metabolites of PCB11 in human blood. Because PCBs and PCB metabolites have been suggested to induce oxidative stress, this study sought to determine whether environmental exposure to PCB11 and its 4-hydroxyl metabolite could induce alterations in steady-state levels of reactive oxygen species (ROS) and cytotoxicity in immortalized human prostate epithelial cells (RWPE-1). This study also examines if antioxidants could protect the cells from PCB11-induced cytotoxicity. Exponentially growing RWPE-1 cells were exposed to PCB11 and its metabolite, 3,3′-dichlorobiphenyl-4-ol (4-OH-PCB11), as well as an airborne PCB mixture resembling the Chicago ambient air congener profile, every day for 5 days. Results showed that 4-OH-PCB11 could significantly induce cell growth suppression and decrease the viability and plating efficiency of RWPE-1 cells. 4-OH-PCB11 also significantly increased steady-state levels of intracellular superoxide, O2 •−, as well as hydroperoxides. Finally, treatment with the combination of polyethylene glycol–conjugated CuZn superoxide dismutase and catalase added 1h after 4-OH-PCB11 exposures, significantly protected RWPE-1 cells from PCB toxicity. The results strongly support the hypothesis that exposure to a hydroxylated metabolite of PCB11 can inhibit cell proliferation and cause cytotoxicity by increasing steady-state levels of ROS. Furthermore, antioxidant treatments following PCBs exposure could significantly mitigate the PCB-induced cytotoxicity in exponentially growing human prostate epithelial cells.
doi:10.1093/toxsci/kft186
PMCID: PMC3829572  PMID: 23997111
4-OH-PCB11; ROS; superoxide; cytotoxicity; antioxidant; oxidative stress.
20.  Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity 
Toxicological Sciences  2013;136(1):166-182.
Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP+-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP+-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP+-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP+, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP+ but not during rotenone or 6-OHDA toxicity.
doi:10.1093/toxsci/kft188
PMCID: PMC3829573  PMID: 23997112
autophagy; apoptosis; Atg5; cathepsins; paraquat; rotenone; MPP+; 6-hydroxydopamine; neurodegeneration; Parkinson’s disease.
21.  Genome-Wide RNAi High-Throughput Screen Identifies Proteins Necessary for the AHR-Dependent Induction of CYP1A1 by 2,3,7,8-Tetrachlorodibenzo-p-dioxin 
Toxicological Sciences  2013;136(1):107-119.
The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.
doi:10.1093/toxsci/kft191
PMCID: PMC3829574  PMID: 23997114
CYP1A1; AHR; RNAi; high-throughput screening; TCDD; esiRNA.
22.  Synchrotron-Based Imaging of Chromium and γ-H2AX Immunostaining in the Duodenum Following Repeated Exposure to Cr(VI) in Drinking Water 
Toxicological Sciences  2014;143(1):16-25.
Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ∼2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. These findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.
doi:10.1093/toxsci/kfu206
PMCID: PMC4274380  PMID: 25352572
hexavalent chromium; Cr(VI); synchrotron; duodenum; carcinogenesis; mode of action; H2AX
23.  An Autonomic Link Between Inhaled Diesel Exhaust and Impaired Cardiac Performance: Insight From Treadmill and Dobutamine Challenges in Heart Failure–Prone Rats 
Toxicological Sciences  2013;135(2):425-436.
Cardiac disease exacerbation is associated with short-term exposure to vehicular emissions. Diesel exhaust (DE) might impair cardiac performance in part through perturbing efferent sympathetic and parasympathetic autonomic nervous system (ANS) input to the heart. We hypothesized that acute changes in ANS balance mediate decreased cardiac performance upon DE inhalation. Young adult heart failure–prone rats were implanted with radiotelemeters to measure heart rate (HR), HR variability (HRV), blood pressure (BP), core body temperature, and pre-ejection period (PEP, a contractility index). Animals pretreated with sympathetic antagonist (atenolol), parasympathetic antagonist (atropine), or saline were exposed to DE (500 µg/m3 fine particulate matter, 4h) or filtered air and then treadmill exercise challenged. At 1 day postexposure, separate rats were catheterized for left ventricular pressure (LVP), contractility, and lusitropy and assessed for autonomic influence using the sympathoagonist dobutamine and surgical vagotomy. During DE exposure, atenolol inhibited increases in HR, BP, and contractility, but not body temperature, suggesting a role for sympathetic dominance. During treadmill recovery at 4h post-DE exposure, HR and HRV indicated parasympathetic dominance in saline- and atenolol-pretreated groups that atropine inhibited. Conversely, at treadmill recovery 21h post-DE exposure, HRV and PEP indicated sympathetic dominance and subsequently diminished contractility that only atenolol inhibited. LVP at 1 day postexposure indicated that DE impaired contractility and lusitropy while abolishing parasympathetic-regulated cardiac responses to dobutamine. This is the first evidence that air pollutant inhalation both causes time-dependent oscillations between sympathetic and parasympathetic dominance and decreases cardiac performance via aberrant sympathetic dominance.
doi:10.1093/toxsci/kft155
PMCID: PMC3937599  PMID: 23872579
air pollution; autonomic; cardiac function; cardiovascular; diesel exhaust; electrocardiography; heart failure; heart rate variability; rat; stress test.
24.  Macrophage Solubilization and Cytotoxicity of Indium-Containing Particles In Vitro  
Toxicological Sciences  2013;135(2):414-424.
Indium-containing particles (ICPs) are used extensively in the microelectronics industry. Pulmonary toxicity is observed after inhalation exposure to ICPs; however, the mechanism(s) of pathogenesis is unclear. ICPs are insoluble at physiological pH and are initially engulfed by alveolar macrophages (and likely airway epithelial cells). We hypothesized that uptake of ICPs by macrophages followed by phagolysosomal acidification results in the solubilization of ICPs into cytotoxic indium ions. To address this, we characterized the in vitro cytotoxicity of indium phosphide (InP) or indium tin oxide (ITO) particles with macrophages (RAW cells) and lung-derived epithelial (LA-4) cells at 24h using metabolic (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) and membrane integrity (lactate dehydrogenase) assays. InP and ITO were readily phagocytosed by RAW and LA-4 cells; however, the particles were much more cytotoxic to RAW cells and cytotoxicity was dose dependent. Treatment of RAW cells with cytochalasin D (CytoD) blocked particle phagocytosis and reduced cytotoxicity. Treatment of RAW cells with bafilomycin A1, a specific inhibitor of phagolysosomal acidification, also reduced cytotoxicity but did not block particle uptake. Based on direct indium measurements, the concentration of ionic indium was increased in culture medium from RAW but not LA-4 cells following 24-h treatment with particles. Ionic indium derived from RAW cells was significantly reduced by treatment with CytoD. These data implicate macrophage uptake and solubilization of InP and ITO via phagolysosomal acidification as requisite for particle-induced cytotoxicity and the release of indium ions. This may apply to other ICPs and strongly supports the notion that ICPs require solubilization in order to be toxic.
doi:10.1093/toxsci/kft154
PMCID: PMC3807620  PMID: 23872580
indium; InP; ITO; solubilization; macrophage cytotoxicity.
25.  Toxicogenomic Evaluation of Long-term Hepatic Effects of TCDD in Immature, Ovariectomized C57BL/6 Mice 
Toxicological Sciences  2013;135(2):465-475.
Acute exposure to hepatotoxic doses of 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD) in mice is characterized by differential gene expression that can be phenotypically anchored to elevated levels of serum alanine aminotransferase, increased relative liver weights, hepatic steatosis, inflammation, and hepatocellular necrosis. Unlike most studies that focus on acute exposure effects, this study evaluated the long-term effects of a single oral gavage of 30 μg/kg TCDD at 1, 4, 12, 24, 36, and 72 weeks postdose in ovariectomized C57BL/6 mice. Hepatic TCDD levels were almost completely eliminated by 24 weeks with a calculated half-life of 12 days. Hepatic gene expression analysis identified 395 unique differentially expressed genes between 1 and 12 weeks that decreased to ≤ 8 by 72 weeks, consistent with the minimal hepatic TCDD levels. Hepatic vacuolization, characteristic of short-term exposure, subsided by 4 weeks. Similarly, TCDD-elicited hepatic necrosis and inflammation dissipated by 1 week. Collectively, these results suggest that TCDD-elicited histologic and gene expression responses can be correlated to elevated hepatic TCDD levels, which, once eliminated, elicit minimal hepatic gene expression and histologic alterations.
doi:10.1093/toxsci/kft156
PMCID: PMC3807621  PMID: 23864506
TCDD; long term; liver; microarrays; mouse.

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