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1.  In vitro assessment of human nuclear hormone receptor activity and cytotoxicity of the flame retardant mixture FM 550 and its triarylphosphate and brominated components 
Toxicology letters  2014;228(2):93-102.
Firemaster® 550 (FM 550) is a mixture of brominated and triarylphosphate flame retardants used in polyurethane foam-based products. The primary components are also used in numerous other applications and are thus common household and industrial contaminants. Our previous animal studies suggested that FM 550 exposure may alter metabolism and cause weight gain. Employing human nuclear receptor (NR) luciferase reporter assays, the goal of this study was to evaluate the agonist actions of FM 550 and its constituent compounds at NRs with known roles in establishing or regulating energy balance. FM 550 was found to have significant agonist activity only at the master regulator of adipocyte differentiation PPARγ. As a result, the concentration response relationships and relative activities of FM 550 at PPARγ were investigated in more detail with the contribution of each chemical component defined and compared to the activities of the prototypical PPARγ environmental ligands triphenyltin and tributylytin. The resulting data indicated that the primary metabolic disruptive effects of FM 550 were likely mediated by the activity of the triarylphosphates at PPARγ, and have identified TPP as a candidate metabolic disruptor that also acts as a cytotoxicant.
PMCID: PMC4180444  PMID: 24786373
apoptosis; endocrine disruptor; flame retardant; nuclear receptors; obesity; peroxisome proliferator-activated receptor
Toxicology letters  2014;228(1):56-66.
Lithocholic acid (LCA) supplementation in the diet results in intrahepatic cholestasis and bile infarcts. Previously we showed that an innate immune response is critical for cholestatic liver injury in the bile duct ligated mice. Thus, the purpose of this study was to investigate the role of neutrophils in the mechanism of liver injury caused by feeding mice a diet containing LCA. C57BL/6 mice were given control or 1% LCA containing diet for 24–96h and then examined for parameters of hepatotoxicity. Plasma ALT levels were significantly increased by 48h after LCA feeding, which correlated with both neutrophil recruitment to the liver and upregulation of numerous pro-inflammatory genes. The injury was confirmed by histology. Deficiency in intercellular adhesion molecule-1 (ICAM-1) expression or inhibition of neutrophil function failed to protect against the injury. Bile acid levels were quantified in plasma and bile of LCA-fed mice after 48 and 96h. Only the observed biliary levels of taurochenodeoxycholic acid and potentially tauro-LCA caused direct cytotoxicity in mouse hepatocytes. These data support the conclusion that neutrophil recruitment occurs after the onset of bile acid-induced necrosis in LCA-fed animals, and is not a primary mechanism of cell death when cholestasis occurs through accumulation of hydrophobic bile acids.
PMCID: PMC4057375  PMID: 24742700
bile acid hepatotoxicity; lithocholic acid; cholestasis; innate immunity; neutrophils
3.  Effects of environmental levels of cadmium, lead and mercury on human renal function evaluated by structural equation modeling 
Toxicology letters  2014;228(1):34-41.
A relationship between exposure to heavy metals, including lead and cadmium, and renal dysfunction has long been suggested. However, modeling of the potential additive, or synergistic, impact of metals on renal dysfunction has proven to be challenging. In these studies, we used structural equation modeling (SEM), to investigate the relationship between heavy metal burden (serum and urine levels of lead, cadmium and mercury) and renal function using data from the NHANES database. We were able to generate a model with goodness of fit indices consistent with a well-fitting model. This model demonstrated that lead and cadmium had a negative relationship with renal function, while mercury did not contribute to renal dysfunction. Interestingly, a linear relationship between lead and loss of renal function was observed, while the maximal impact of cadmium occurred at or above serum cadmium levels of 0.8 µg/L. The interaction of lead and cadmium in loss of renal function was also observed in the model. These data highlight the use of SEM to model interaction between environmental contaminants and pathophysiology, which has important implications in mechanistic and regulatory toxicology.
PMCID: PMC4084726  PMID: 24769258
Cadmium; CDC; Centers for Disease Control and Prevention; Kidney Function; Lead; Mercury; National Health and Nutrition Examination Survey; NHANES; SEM; Structural Equation Model
4.  In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes 
Toxicology letters  2014;228(1):48-55.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (VMAX, nmol/min/mg microsomal protein), affinity constants (KM, μM), and rates of intrinsic clearance (CLINT, ml/min/kg body weight) were calculated from substrate depletion data. CLINT was also estimated from substrate depletion data using the alternative in vitro half-life method. VMAX and CLINT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored.
PMCID: PMC4274170  PMID: 24769260
Benzo[a]pyrene; Dibenzo[def,p]chrysene; Michaelis–Menten; VMAX; KM; Intrinsic clearance
5.  Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure 
Toxicology letters  2014;227(2):99-112.
A typical clinical cis-diamminedichloroplatinum (II) (cisplatin) dosing regimen consists of repeated treatment cycles followed by a recovery period. While effective, this dosing structure results in a prolonged, often permanent, infertility in men. Spermatogonial stem cells (SSCs) are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. We propose that an altered spermatogonial environment during recovery from the initial treatment cycle drives an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced injury from subsequent cycles. To test this hypothesis, the undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery periods of a clinically-relevant cisplatin exposure paradigm. Histological examination revealed a disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of Terminal Deoxynucleotidyl Transferase-Mediated Digoxigenin-dUTP Nick End Labeling (TUNEL) staining indicated an increase in apoptotic frequency following exposure. Immunohistochemical examination of Foxo1 and incorporated BrdU showed an increase in the undifferentiated spermatogonial population and mitotic activity in the recovery period in mice exposed to one cycle, but not two cycles of cisplatin. Immunohistochemical investigation of glial cell line-derived neurotrophic factor (GDNF) revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.
PMCID: PMC4041011  PMID: 24704392
cisplatin; GDNF; spermatogonia; SSC niche; Sertoli cell; stem germ cell
6.  Heavy Metal Scavenger Metallothionein Attenuates ER Stress-Induced Myocardial Contractile Anomalies: Role of Autophagy 
Toxicology letters  2014;225(3):333-341.
Endoplasmic reticulum (ER) stress increases the risk of cardiovascular morbidity and mortality although the underlying mechanism remains elusive. This study was designed to examine the impact of cardiac over-expression of metallothionein, a cysteine-rich heavy metal scavenger, on ER stress-induced changes in myocardial function and underlying mechanism involved with a focus on autophagy. Wild-type friendly virus B (FVB) and metallothionein transgenic mice were subjected to the ER stress inducer tunicamycin (1 mg/kg). Our results showed that ER stress led to compromised echocardiographic and cardiomyocyte contractile function, intracellular Ca2+ mishandling. Tunicamycin promoted ER stress and oxidative stress, increased left ventricular end systolic and diastolic diameter, as well as suppressed fractional shortening and whole heart contractility, the effects of which were significantly attenuated or ablated by metallothionein. Levels of the autophagy markers such as phosphorylated ULK1, Atg5, Atg7, LC3B and the autophagy adaptor p62 were significantly upregulated. These ER stress-induced changes in myocardial function, autophagy and autophagy signaling were distinctly mitigated or alleviated by metallothionein. Inhibition of autophagy using 3-methyladenine in vitro reversed ER stress-induced cardiomyocyte contractile defects. Meanwhile, ER stress-induced cardiomyocyte dysfunction was attenuated by the antioxidant N-acetylcysteine. Collectively, these findings suggested that metallothionein protects against ER stress-induced cardiac anomalies possibly through attenuation of cardiac autophagy.
PMCID: PMC4041391  PMID: 24440343
ER stress; Metallothionein; Tunicamycin; Cardiac function; Autophagy
7.  Early chronic low-level lead exposure produces glomerular hypertrophy in young C57BL/6J mice☆ 
Toxicology letters  2013;225(1):48-56.
Early chronic lead exposure continues to pose serious health risks for children, particularly those living in lower socioeconomic environments. This study examined effects on developing glomeruli in young C57BL/6J mice exposed to low (30 ppm), higher (330 ppm) or no lead via dams’ drinking water from birth to sacrifice on post-natal day 28. Low-level lead exposed mice [BLL mean (SD); 3.19 (0.70) μg/dL] had an increase in glomerular volume but no change in podocyte number compared to control mice [0.03 (0.01) μg/dL]. Higher-level lead exposed mice [14.68 (2.74) μg/dL] had no change in either glomerular volume or podocyte number. The increase in glomerular volume was explained by increases in glomerular capillary and mesangial volumes with no change in podocyte volume. Early chronic lead exposure yielding very low blood lead levels alters glomerular development in pre-adolescent animals.
PMCID: PMC3957482  PMID: 24300173
Pb toxicity; Glomerular hypertrophy; Capillary volume; Mesangial volume; Design-based stereology
8.  Exposure to inhaled particulate matter activates early markers of oxidative stress, inflammation and unfolded protein response in rat striatum 
Toxicology letters  2013;222(2):146-154.
To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32 µg/m3), fine (178 µg/m3) or ultrafine (107 µg/m3) concentrated PM or filtered air. Different brain regions (olfactory bulb, frontal cortex, striatum and hippocampus), were harvested from the rats following exposure to airborne PM. Subsequently, prooxidant (HO-1 and SOD-2), and inflammatory markers (IL-1β and TNFα), apoptotic (caspase 3), and unfolded protein response (UPR) markers (XBP-1S and BiP), were also measured using real-time PCR. Activation of nuclear transcription factors Nrf-2 and NF-κB, associated with antioxidant and inflammation processes, respectively, were also analyzed by GSMA. Ultrafine PM increased HO-1 and SOD-2 mRNA levels in the striatum and hippocampus, in the presence of Nrf-2 activation. Also, ultrafine PM activated NF-κB and increased IL-1β and TNFα in the striatum. Activation of UPR was observed after exposure to coarse PM through the increment of XBP-1S and BiP in the striatum, accompanied by an increase in antioxidant response markers HO-1 and SOD-2. Our results indicate that exposure to different size fractions of PM may induce physiological changes (in a neuroanatomical manner) in the central nervous system (CNS), specifically within the striatum, where inflammation, oxidative stress and UPR signals were effectively activated.
PMCID: PMC4318358  PMID: 23892126
Particulate matter; Central nervous system; Striatum; Oxidative stress; Inflammation; Unfolded protein response
9.  [No title available] 
PMCID: PMC3951739  PMID: 24270004
10.  [No title available] 
PMCID: PMC3987699  PMID: 24275386
11.  Motor neuron disease due to neuropathy target esterase mutation: enzyme analysis of fibroblasts from human subjects yields insights into pathogenesis 
Toxicology letters  2010;199(1):1-5.
Recently, we identified neuropathy target esterase (NTE) mutation as the cause of an autosomal recessive motor neuron disease (NTE-MND). Subsequently, we showed that NTE-MND mutations reduced specific activity (SA) and altered inhibitory kinetics of NTE catalytic domain constructs. Recent preliminary results showed that NTE is expressed in cultured human skin fibroblasts, and others have used mutant forms of neuronal proteins expressed in fibroblasts as biomarkers of neurogenetic diseases. Therefore, the present study was carried out to test the hypothesis that NTE in cultured skin fibroblasts from NTE-MND subjects also exhibit altered enzymological properties assessed by SA and IC50 values of mipafox (MIP) and chlorpyrifos oxon (CPO). NTE SA was reduced to 65% of control (wild type NTE from commercially obtained fibroblasts) in homozygous M1012V fibroblasts and 59-61% of control in compound heterozygous R890H/c2946_2947InsCAGC fibroblasts. MIP IC50 values were unaffected by the NTE mutations, but the CPO IC50 increased 4.5-fold in homozygous M1012V fibroblasts. Interestingly, markedly reduced NTE SAs (40-43% of control) were observed in fibroblasts from asymptomatic subjects heterozygous for NTE insertion c2946_2947InsCAGC. This insertion is predicted to produce truncated NTE missing the last 235 residues of its catalytic domain. These observations confirm that NTE-MND mutations reduce NTE SA in vitro. Moreover, to the extent observations made in cultured fibroblasts may be generalized to events in the nervous system, lack of correlation between reduced fibroblast NTE SA and the occurrence of NTE-MND in NTE insertion mutation heterozygotes indicates that reduction of NTE SA alone is insufficient to cause MND.
PMCID: PMC4310456  PMID: 20603202
Fibroblast; Motor neuron disease (MND); Mutation; Neuropathy target esterase (NTE); Organophosphorus compound-induced delayed neurotoxicity (OPIDN)
12.  Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties 
Toxicology letters  2010;196(2):67-73.
Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (ki) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.
PMCID: PMC4310459  PMID: 20382209
Hereditary spastic paraplegia (HSP); motor neuron disease (MND); neuropathy target esterase (NTE); organophosphorus compound-induced delayed neuropathy (OPIDN)
Toxicology letters  2013;224(2):10.1016/j.toxlet.2013.10.030.
Osteopontin (OPN) is a chemotactic factor which can be cleaved to the pro-inflammatory form by matrix metalloproteinases (MMPs). To test the hypothesis that OPN can modulate inflammatory liver injury during cholestasis, wild-type (WT) C57BL/6 and OPN knockout (OPN-KO) mice underwent bile duct ligation (BDL). OPN-KO mice showed significant reduction in liver injury (plasma ALT and necrosis) and neutrophil recruitment compared with WT animals at 24h but not 72h after BDL. In WT mice, a 4-fold increase in hepatic MMP-3 mRNA and elevated MMP activities and cleaved OPN levels were observed in bile. WT mice subjected to BDL in the presence of the MMP inhibitor BB-94 showed reduced liver injury, less neutrophil extravasation and diminished levels of cleaved OPN in bile. Thus, during obstructive cholestasis, OPN released from biliary epithelial cells could be cleaved by MMPs in bile. When the biliary system leaks, cleaved OPN enters the parenchyma and attracts neutrophils. In the absence of OPN, other chemoattractants, e.g. chemokines, mediate a delayed inflammatory response and injury. Taken together, our data suggest that OPN is the pro-inflammatory mediator that initiates the early neutrophil-mediated injury phase during obstructive cholestasis in mice.
PMCID: PMC3875222  PMID: 24188933
Inflammatory Liver Injury; Cholestasis; Bile duct ligation; Osteopontin; Neutrophils
14.  Formaldehyde Induces Micronuclei in Mouse Erythropoietic Cells and Suppresses the Expansion of Human Erythroid Progenitor Cells 
Toxicology letters  2013;224(2):233-239.
Although formaldehyde (FA) has been classified as a human leukemogen, the mechanisms of leukemogenesis remain elusive. Previously, using colony-forming assays in semi-solid media, we showed that FA exposure in vivo and in vitro was toxic to human hematopoietic stem/progenitor cells. In the present study, we have applied new liquid in vitro erythroid expansion systems to further investigate the toxic effects of FA (0–150 µM) on cultured mouse and human hematopoietic stem/progenitor cells. We determined micronucleus (MN) levels in polychromatic erythrocytes (PCEs) differentiated from mouse bone marrow. We measured cell growth, cell cycle distribution, and chromosomal instability, in erythroid progenitor cells (EPCs) expanded from human peripheral blood mononuclear cells. FA significantly induced MN in mouse PCEs and suppressed human EPC expansion in a dose-dependent manner, compared with untreated controls. In the expanded human EPCs, FA slightly increased the proportion of cells in G2/M at 100 µM and aneuploidy frequency in chromosomes 7 and 8 at 50 µM. Our findings provide further evidence of the toxicity of FA to hematopoietic stem/progenitor cells and support the biological plausibility of FA-induced leukemogenesis.
PMCID: PMC3891867  PMID: 24188930
formaldehyde; erythroid progenitor; micronuclei; aneuploidy
15.  Differences in Butadiene Adduct Formation between Rats and Mice Not Due to Selective Inhibition of CYP2E1 by Butadiene Metabolites 
Toxicology letters  2013;223(2):10.1016/j.toxlet.2013.08.025.
CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increase linearly at low BD exposures and then saturate at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54 μM, mouse; 98 μM, rat), BD-diol considerably weaker (IC50 1200 μM, mouse; 1000 μM, rat), and DEB inhibition nonexistent (IC50 >25 mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis.
PMCID: PMC3831829  PMID: 24021170
CYP2E1; butadiene; cooperativity; inactivation; mouse; rat
16.  In Utero Exposure to Benzo[a]pyrene Increases Adiposity and Causes Hepatic Steatosis in Female Mice, and Glutathione Deficiency Is Protective 
Toxicology letters  2013;223(2):10.1016/j.toxlet.2013.09.017.
Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Reactive metabolites and reactive oxygen species generated during PAH metabolism are detoxified by reactions involving glutathione (GSH). Early life exposures to tobacco smoke and air pollution have been linked to increased risk of obesity and metabolic syndrome. We investigated the independent and interactive effects of prenatal exposure to BaP and GSH deficiency due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, on adiposity and hepatic steatosis in adult female F1 offspring. We mated Gclm+/- dams with Gclm+/- males and treated the pregnant dams with 0, 2, or 10 mg/kg/day BaP in sesame oil by oral gavage daily from gestational day 7 through 16. We analyzed metabolic endpoints in female Gclm-/- and Gclm+/+ littermate F1 offspring. Prenatal BaP exposure significantly increased visceral adipose tissue weight, weight gain between 3 wks and 7.5 months of age, hepatic lipid content measured by oil red O staining, and hepatic fatty acid beta-oxidation gene expression in Gclm+/+, but not in Gclm-/-, female offspring. Hepatic expression of lipid biosynthesis and antioxidant genes were decreased and increased, respectively, in Gclm-/- mice. Our results suggest that reported effects of pre- and peri-natal air pollution and tobacco smoke exposure on obesity may be mediated in part by PAHs. GSH deficiency is protective against the metabolic effects of prenatal BaP exposure.
PMCID: PMC3856224  PMID: 24107266
polycyclic aromatic hydrocarbons; glutathione; fatty liver; glutamate cysteine ligase; obesity; prenatal programming
17.  Alcoholic Lung Injury: Metabolic, Biochemical and Immunological Aspects 
Toxicology letters  2013;222(2):10.1016/j.toxlet.2013.07.016.
Chronic alcohol abuse is a systemic disorder and a risk factor for acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). A significant amount of ingested alcohol reaches airway passages in the lungs and can be metabolized via oxidative and non-oxidative pathways. About 90% of the ingested alcohol is metabolized via hepatic alcohol dehydrogenase (ADH)-catalyzed oxidative pathway. Alcohol can also be metabolized by cytochrome P450 2E1 (CYP2E1), particularly during chronic alcohol abuse. Both the oxidative pathways, however, are associated with oxidative stress due to the formation of acetaldehyde and/or reactive oxygen species (ROS). Alcohol ingestion is also known to cause endoplasmic reticulum (ER) stress, which can be mediated by oxidative and/or non-oxidative metabolites of ethanol. An acute as well as chronic alcohol ingestions impair protective antioxidants, oxidize reduced glutathione (GSH, cellular antioxidant against ROS and oxidative stress), and suppress innate and adaptive immunity in the lungs. Oxidative stress and suppressed immunity in the lungs of chronic alcohol abusers collectively are considered to be major risk factors for infection and development of pneumonia, and such diseases as ARDS and COPD. Prior human and experimental studies attempted to identify common mechanisms by which alcohol abuse directly causes toxicity to alveolar epithelium and respiratory tract, particularly lungs. In this review, the metabolic basis of lung injury, oxidative and ER stress and immunosuppression in experimental models and alcoholic patients, as well as potential immunomodulatory therapeutic strategies for improving host defenses against alcohol-induced pulmonary infections are discussed.
PMCID: PMC3806189  PMID: 23892124
18.  Methylmercury exposure increases lipocalin related (lpr) and decreases activated in blocked unfolded protein response (abu) genes and specific miRNAs in Caenorhabditis elegans 
Toxicology letters  2013;222(2):10.1016/j.toxlet.2013.07.014.
Methylmercury (MeHg) is a persistent environmental and dietary contaminant that causes serious adverse developmental and physiologic effects at multiple cellular levels. In order to understand more fully the consequences of MeHg exposure at the molecular level, we profiled gene and miRNA transcripts from the model organism Caenorhabditis elegans. Animals were exposed to MeHg (10µM) from embryo to larval 4 (L4) stage and RNAs were isolated. RNA-seq analysis on the Illumina platform revealed 541 genes up- and 261 genes down-regulated at a cutoff of 2-fold change and false discovery rate-corrected significance q < 0.05. Among the up-regulated genes were those previously shown to increase under oxidative stress conditions including hsp-16.11 (2.5-fold), gst-35 (10.1-fold), and fmo-2(58.5-fold). In addition, we observed up-regulation of 6 out of 7 lipocalin related (lpr) family genes and down regulation of 7 out of 15 activated in blocked unfolded protein response (abu) genes. Gene Ontology enrichment analysis highlighted the effect of genes related to development and organism growth. miRNA-seq analysis revealed 6–8 fold down regulation of mir-37-3p, mir-41-5p, mir-70-3p, and mir-75-3p. Our results demonstrate the effects of MeHg on specific transcripts encoding proteins in oxidative stress responses and in ER stress pathways. Pending confirmation of these transcript changes at protein levels, their association and dissocation characteristics with interaction partners, and integration of these signals, these findings indicate broad and dynamic mechanisms by which MeHg exerts its harmful effects.
PMCID: PMC3816353  PMID: 23872261
heavy metal; oxidative stress; ER stress; next-generation sequencing; blocked unfolded protein response
19.  Comparisons of differential gene expression elicited by TCDD, PCB126, βNF, or ICZ in mouse hepatoma Hepa1c1c7 cells and C57BL/6 mouse liver 
Toxicology letters  2013;223(1):52-59.
The aryl hydrocarbon receptor (AhR) is a promiscuous receptor activated by structurally diverse synthetic and natural compounds. AhR activation may lead to ligand-specific changes in gene expression despite similarities in mode of action. Therefore, differential gene expression elicited by four structurally diverse, high affinity AhR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM, 30 µg/kg), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126; 100 nM, 300 µg/kg), β-naphthoflavone (βNF; 10 µM, 90 mg/kg), and indolo[3,2-b]carbazole (ICZ; 1 µM)) in mouse Hepa1c1c7 hepatoma cells and C57BL/6 mouse liver samples were compared. A total of 288, 183, 119, and 131 Hepa1c1c7 genes were differentially expressed (|fold-change| ≥ 1.5, P1(t) ≥ 0.9999) by TCDD, βNF, PCB126, and ICZ, respectively. Only ~35% were differentially expressed by all 4 ligands in Hepa1c1c7 cells. In vivo, 661, 479, and 265 hepatic genes were differentially expressed following treatment with TCDD, βNF, and PCB126, respectively. Similar to Hepa1c1c7 cells, ≤34% of gene expression changes were common across all ligands. Principal components analysis identified time-dependent gene expression divergence. Comparisons of ligand-elicited expression between Hepa1c1c7 cells and mouse liver identified only 11 common gene expression changes across all ligands. Although metabolism may explain some ligand-specific gene expression changes, PCB126, βNF, and ICZ also elicited divergent expression compared to TCDD, suggestive of selective AhR modulation.
PMCID: PMC4096832  PMID: 23994337
Selective AhR modulation; Microarray; Mouse; Cross-ligand comparison
20.  Chronic Manganese Exposure Impairs Visuospatial Associative Learning in Non-Human Primates 
Toxicology letters  2013;221(2):146-151.
Manganese (Mn) is an essential trace metal nutrient, however, excess Mn can be neurotoxic. The degree to which chronic environmental or occupational exposures to Mn in adults cause neuropsychological dysfunction is of considerable interest. Descriptions of neuropsychological dysfunction following chronic Mn exposure have been somewhat inconsistent though, likely owing to different measures of exposure in different populations, complicated by factors of mixed exposures and differences in neuropsychological tests administered. We previously described up-regulation of the mRNA expression for amyloid-beta (A-beta) precursor-like protein 1 (APLP1) and the presence of A-beta diffuse plaques in frontal cortex of Mn-exposed monkeys. The present study examined Mn-induced changes in performance on a paired associate learning (PAL) task that has been suggested as a marker for preclinical Alzheimer’s disease. Aspects of performance of this task were affected early following initiation of Mn exposure. Thus, PAL performance may be a sensitive and valuable tool for the early, preclinical detection of incipient dementia and it may also be a sensitive tool for detecting cognitive dysfunction from Mn exposure. The current cognitive data, combined with our previous findings, suggest that frontal cortex may be a particularly sensitive target for the effects of Mn on cognition and that chronic Mn exposure may initiate or accelerate a process that could lead to or predispose to Alzheimer’s like pathology and cognitive dysfunction.
PMCID: PMC3864566  PMID: 23778301
manganese; cognition; monkeys
21.  Comparison of 2, 2-Bis (bromomethyl)-1, 3-propanediol induced genotoxcity in UROtsa cells and primary rat hepatocytes: relevance of metabolism and oxidative stress 
Toxicology letters  2013;222(3):273-279.
2, 2-Bis (bromomethyl)-1, 3-propanediol (BMP) is a brominated flame retardant used in urethane foams and polyester resins. In a two year dietary study, BMP caused neoplastic lesions at multiple sites including the urinary bladder of both rats and mice. However, liver was not a target tissue. We previously reported that BMP elicited oxidative DNA damage in a human uroepithelial cell line (UROtsa). The present in vitro study investigated the susceptibility of target (UROtsa cells) and non-target cells (primary rat hepatocytes) to BMP-induced genotoxicity. In contrast to hepatocytes, BMP exhibited greater genotoxic potential in UROtsa cells as evidenced by the concentration dependent increase in DNA strand breaks and DNA binding. Total content of intracellular GSH quantified in UROtsa cells (2.7 ± 1.0 nmol/mg protein) was 4 fold lower than that in hepatocytes (10.7 ± 0.3 nmol/mg protein). HPLC analysis indicated BMP was not metabolized and/or consumed in UROtsa cells at any of the concentrations tested (10–250 µM) but was extensively converted to a mono-glucuronide in hepatocytes. These results demonstrate that a target cell line such as UROtsa cells are more susceptible to BMP-induced DNA damage when compared to non-target cells. This increased susceptibility may relate to the deficiency of antioxidant and/or metabolic capabilities in UROtsa cells.
PMCID: PMC3804725  PMID: 23954263
2, 2-Bis (bromomethyl)-1, 3-propanediol; UROtsa cell; primary rat hepatocyte, cell susceptibility; metabolism; oxidative stress
22.  Structure–activity relationships for perfluoroalkane-induced in vitro interference with rat liver mitochondrial respiration✩ 
Toxicology letters  2013;222(3):257-264.
Perfluorinated alkyl acids (PFAAs) represent a broad class of commercial products designed primarily for the coatings industry. However, detection of residues globally in a variety of species led to the discontinuation of production in the U.S. Although PFAAs cause activation of the PPARα and CAR nuclear receptors, interference with mitochondrial bioenergetics has been implicated as an alternative mechanism of cytotoxicity. Although the mechanisms by which the eight carbon chain PFAAs interfere with mitochondrial bioenergetics are fairly well described, the activities of the more highly substituted or shorter chain PFAAs are far less well characterized. The current investigation was designed to explore structure–activity relationships by which PFAAs interfere with mitochondrial respiration in vitro. Freshly isolated rat liver mitochondria were incubated with one of 16 different PFAAs, including perfluorinated carboxylic, acetic, and sulfonic acids, sulfonamides and sulfamido acetates, and alcohols. The effect on mitochondrial respiration was measured at five concentrations and dose–response curves were generated to describe the effects on state 3 and 4 respiration and respiratory control. With the exception of PFOS, all PFAAs at sufficiently high concentrations (>20 μM) stimulated state 4 and inhibited state 3 respiration. Stimulation of state 4 respiration was most pronounced for the carboxylic acids and the sulfonamides, which supports prior evidence that the perfluorinated carboxylic and acetic acids induce the mitochondrial permeability transition, whereas the sulfonamides are protonophoric uncouplers of oxidative phosphorylation. In both cases, potency increased with increasing carbon number, with a prominent inflection point between the six and eight carbon congeners. The results provide a foundation for classifying PFAAs according to specific modes of mitochondrial activity and, in combination with toxicokinetic considerations, establishing structure–activity-based boundaries for initial estimates of risk for noncancer endpoints for PFAAs for which minimal in vivo toxicity testing currently exists.
PMCID: PMC3935505  PMID: 23954199
Perfluoroalkyl acids (PFAA); Mitochondria; Structure-activity
23.  Differential immunotoxic effects of inorganic and organic mercury species in vitro 
Toxicology letters  2010;198(2):182-190.
Despite the fact that humans are exposed to multiple forms of mercury (elemental, inorganic, and organic), most research on mercury toxicity has focused on methylmercury (MeHg) and on neurotoxic outcomes and mechanisms. Recent work has indicated that the immunotoxic effects of mercury compounds may be significant contributors to human disease as well as mechanistically relevant to other target organ toxicities. In this study, we compared the effects of inorganic Hg (iHg) to organic Hg species (MeHg and ethylmercury, EtHg) in human peripheral blood mononuclear cells (PBMCs) in vitro at sub -cytotoxic concentrations, using methods developed to characterize response of human PBMCs to iHg in vitro. PBMCs were isolated from six volunteer blood donors (3 males, 3 females) and cultured in the presence and absence of lipopolysaccharide (LPS) and low levels (up to 200 nM of each Hg species, separately) for 24 hours in culture. Cell culture supernatants were analyzed for cytokine concentrations with a bead-based multiplex assay.
We report that iHg and MeHg both increase pro-inflammatory cytokine release in LPS-stimulated PBMCs, while EtHg decreases IFN-γ release as well pro-inflammatory cytokine release. IL-17 release is significantly increased only in response to iHg treatment. Levels of anti-inflammatory cytokines (IL-1Ra and IL-10) were not significantly altered by any Hg treatment. These results indicate that both organic and inorganic species of Hg can affect the human immune system, but that they may exert different effects on immune function.
PMCID: PMC4160064  PMID: 20600710
mercury; immunotoxicity; humans; in vitro; cytokines
24.  Perinatal exposure to polychlorinated biphenyls alters social behaviors in rats 
Toxicology letters  2010;199(2):136-143.
Perinatal exposure to polychlorinated biphenyls (PCBs) leads to significant alterations of neural and hormonal systems. These alterations have been shown to impair motor and sensory development. Less is known about the influence of PCB exposure on developing emotional and motivational systems involved in social interactions and social learning. The present study examined the impact of perinatal PCB exposure (mixture of congeners 47 and 77) on social recognition in juvenile animals, conspecific-directed investigation in adults and on neural and hormonal systems involved in social functions. We used a standard habituation–dishabituation paradigm to evaluate juvenile recognition and a social port paradigm to monitor adult social investigation. Areal measures of the periventricular nucleus (PVN) of the hypothalamus were obtained to provide correlations with related hormone and brain systems. PCB exposed rats were significantly impaired in social recognition as indicated by persistent conspecific-directed exploration by juvenile animals regardless of social experience. As adults, PCB exposure led to a dampening of the isolation-induced enhancement of social investigation. There was not a concomitant alteration of social investigation in pair-housed PCB exposed animals at this stage of development. Interestingly, PVN area was significantly decreased in juvenile animals exposed to PCB during the perinatal period. Shifts in hypothalamic regulation of hormones involved in social behavior and stress could be involved in the behavioral changes observed. Overall, the results suggest that PCB exposure impairs context or experience-dependent modulation of social approach and investigation. These types of social-context deficits are similar to behavioral deficits observed in social disorders such as autism and other pervasive developmental disorders.
PMCID: PMC4139010  PMID: 20813172
Affect; Emotion; Development; Hormones; Motivation; Toxicant
25.  Involvement of CTR1 and ATP7A in lead (Pb)-induced copper (Cu) accumulation in choroidal epithelial cells 
Toxicology letters  2013;225(1):110-118.
The blood–cerebrospinal fluid barrier (BCB) plays a key role in maintaining copper (Cu) homeostasis in the brain. Cumulative evidences indicate that lead (Pb) exposure alters cerebral Cu homeostasis, which may underlie the development of neurodegenerative diseases. This study investigated the roles of Cu transporter 1 (CTR1) and ATP7A, two Cu transporters, in Pb-induced Cu accumulation in the choroidal epithelial cells. Pb exposure resulted in increased intracellular 64Cu retention, accompanying with up-regulated CTR1 level. Knockdown of CTR1 using siRNA before Pb exposure diminished the Pb-induced increase of 64Cu uptake. The expression level of ATP7A was down-regulated following the Pb exposure. ATP7A siRNA knockdown, or PCMB treatment, inhibited the 64Cu efflux from the cells, while the following additional incubation with Pb failed to further increase the intracellular 64Cu retention. Cu exposure, or intracellular Cu accumulation following the tetracycline (Tet)-induced overexpression of CTR1, did not result in significant change in ATP7A expression. Taken together, these data indicate that CTR1 and ATP7A play important roles in Cu transport in choroidal epithelial cells, and the Pb-induced intracellular Cu accumulation appears to be mediated, at least in part, via the alteration of CTR1 and ATP7A expression levels following Pb exposure.
PMCID: PMC4127571  PMID: 24316150
Blood–cerebrospinal fluid barrier; Lead; Copper; Copper transporter 1; ATP7A

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