The potential for human exposure to the diverse and ever-changing world of nanoscale materials has raised concerns about their influence on health and disease. The novel physical and chemical properties of these materials, which are associated with their small size, complicate toxicological evaluations. Further, these properties may make engineered nanomaterials (ENMs) a prime target for interaction with the immune system following uptake by phagocytes. Undesired effects on antigen-presenting cells and other phagocytic cells are of concern due to the high likelihood of ENM uptake by these cells. In addition, ENM interactions with lymphocytes and other cell types can contribute to a varied spectrum of possible effects, including inflammation, hypersensitivity, and immunomodulation. Furthermore, the mast cell (a type of immune cell traditionally associated with allergy) appears to contribute to certain inflammatory and toxic effects associated with some ENMs. Although incidental exposure may be undesirable, nanomedicines engineered for various clinical applications provide opportunities to develop therapies that may or may not intentionally target the immune system. The interaction between ENMs and the immune system and the resulting pharmacokinetic and phenotypic responses are critical factors that dictate the balance between toxicity and clinical efficacy of nanotherapeutics.
Bisphenol A (BPA) is a widely used material known to cause adverse effects in humans and other mammals. To date, little is known about the global metabolomic alterations caused by BPA using urinalysis. Sprague-Dawley rats were orally administrated BPA at the levels of 0, 0.5 μg/kg/day and 50 mg/kg/day covering a low dose and a reference dose for 8 weeks. We conducted a capillary electrophoresis in tandem with electrospray ionization time-of-flight mass spectrometry based nontargeted metabolomic analysis using rat urine. To verify the metabolic alteration at both low and high doses, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were further conducted to analyze hepatic expression of methionine adenosyltransferase Iα (Mat1a) and methionine adenosyltransferase IIα (Mat2a). Hepatic S-adenosylmethionine (SAMe) was also analyzed. A total of 199 metabolites were profiled. Statistical analysis and pathway mapping indicated that the most significant metabolic perturbations induced by BPA were the increased biotin and riboflavin excretion, increased synthesis of methylated products, elevated purine nucleotide catabolism, and increased flux through the choline metabolism pathway. We found significantly higher mRNA and protein levels of Mat1a and Mat2a, and significantly higher SAMe levels in rat liver at both low and high doses. These two genes encode critical isoenzymes that catalyze the formation of SAMe, the principal biological methyl donor involved in the choline metabolism. In conclusion, an elevated choline metabolism is underlying the mechanism of highly methylated environment and related metabolic alterations caused by BPA. The data of BPA-elevated accepted biomarkers of injury indicate that BPA induces DNA methylation damage and broad protein degradation, and the increased deleterious metabolites in choline pathway may also be involved in the toxicity of BPA.
Inorganic arsenic (iAs), a human carcinogen, potentially targets the prostate. iAs malignantly transforms the RWPE-1 human prostate epithelial line to CAsE-PE cells, and a derivative normal stem cell (SC) line, WPE-stem, to As-Cancer SC (As-CSC) line. MicroRNAs (miRNA) are noncoding but exert negative control on expression by degradation or translational repression of target mRNAs. Aberrant miRNA expression is important in carcinogenesis. A miRNA array of CAsE-PE and As-CSC revealed common altered expression in both for pathways concerning oncogenesis, miRNA biogenesis, cell signaling, proliferation, and tumor metastasis and invasion. The KRAS oncogene is overexpressed in CAsE-PE cells but not by mutation or promoter hypomethylation, and is intensely overexpressed in As-CSC cells. In both transformants, decreased miRNAs targeting KRAS and RAS superfamily members occurred. Reduced miR-134, miR-373, miR-155, miR-138, miR-205, miR-181d, miR-181c, and let-7 in CAsE-PE cells correlated with increased target RAS oncogenes, RAN, RAB27A, RAB22A mRNAs, and KRAS protein. Reduced miR-143, miR-34c-5p, and miR-205 in As-CSC correlated with increased target RAN mRNA, and KRAS, NRAS, and RRAS proteins. The RAS/ERK and PI3K/PTEN/AKT pathways control cell survival, differentiation, and proliferation, and when dysregulated promote a cancer phenotype. iAs transformation increased expression of activated ERK kinase in both transformants and altered components of the PI3K/PTEN/AKT pathway including decreased PTEN and increases in BCL2, BCL-XL, and VEGF in the absence of AKT activation. Thus, dysregulated miRNA expression may be linked to RAS activation in both transformants.
Cereal grain contamination by trichothecene mycotoxins is known to negatively impact human and animal health with adverse effects on food intake and growth being of particular concern. The head blight fungus Fusarium graminearum elaborates five closely related 8-ketotrichothecene congeners: (1) deoxynivalenol (DON), (2) 3-acetyldeoxynivalenol (3-ADON), (3) 15-acetyldeoxynivalenol (15-ADON), (4) fusarenon X (FX), and (5) nivalenol (NIV). While anorexia induction in mice exposed intraperitoneally to DON has been linked to plasma elevation of the satiety hormones cholecystokinin (CCK) and peptide YY3–36 (PYY3–36), the effects of oral gavage of DON or of other 8-keotrichothecenes on release of these gut peptides have not been established. The purpose of this study was to (1) compare the anorectic responses to the aforementioned 8-ketotrichothecenes following oral gavage at a common dose (2.5 mg/kg bw) and (2) relate these effects to changes plasma CCK and PYY3–36 concentrations. Elevation of plasma CCK markedly corresponded to anorexia induction by DON and all other 8-ketotrichothecenes tested. Furthermore, the CCK1 receptor antagonist SR 27897 and the CCK2 receptor antagonist L-365,260 dose-dependently attenuated both CCK- and DON-induced anorexia, which was consistent with this gut satiety hormone being an important mediator of 8-ketotrichothecene-induced food refusal. In contrast to CCK, PYY3–36 was moderately elevated by oral gavage with DON and NIV but not by 3-ADON, 15-ADON, or FX. Taken together, the results suggest that CCK plays a major role in anorexia induction following oral exposure to 8-ketotrichothecenes, whereas PYY3–36 might play a lesser, congener-dependent role in this response.
To elucidate molecular mechanisms by which the phenolic herbicide ioxynil (IOX) and the brominated flame retardant tetrabromobisphenol A (TBBPA) exert thyroid hormone (TH) disrupting activity, we investigated the effects of the chemicals on the histone and RNA polymerase II (RNAPII) modifications in Xenopus laevis XL58-TRE-Luc cells in direct TH-response genes encoding TH receptor β (Thrb) and TH-induced basic leucine zipper protein (Thibz) using chromatin immunoprecipitation assays. For both the thrb and thibz genes, 3,3',5-triiodothyronine (T3) enhanced the amounts of gene transcripts and increased the amounts of acetylated histone H4 (H4Ac), trimethylated histone H3 lysine 4 (H3K4me3) and phosphorylated RNAPII serine 5 (RNAPIIS5P), epigenetic markers of gene activation at 5' regulatory regions, and the amounts of trimethylated histone H3 lysine 36 (H3K36me3) and phosphorylated RNAPII serine 2 (RNAPIIS2P), epigenetic markers of activation of transcriptional elongation at protein coding regions. Treatment with IOX and TBBPA reduced the amounts of the thrb transcript and suppressed the T3-induced modifications of H3K4me3, RNAPIIS5P, H3K36me3, and RNAPIIS2P. In the thibz gene, IOX and TBBPA did not suppress the T3-induced histone and RNAPII modifications except for H3K36me3 in the TBBPA treatment, despite both chemicals decreasing the T3-induced transcription. Our results demonstrate that IOX and TBBPA affect TH-induced histone and RNAPII modifications, which are involved in early and progressive stages of RNAPII transcriptional elongation, in direct TH-response genes, in somewhat target gene-dependent and chemical-specific manners. Both IOX and TBBPA are likely to influence epigenetically a cascade of TH receptor-mediated gene regulation.
Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by European Centre for the Validation of Alternative Methods (ECVAM). Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-h period. The specificity, the sensitivity, and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the micronucleus (MN) test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.
Increasing evidence demonstrates that cadmium (Cd) induces inflammation, but its mechanisms remain obscure. The present study showed that treatment with CdCl2 selectively upregulates macrophage inflammatory protein (MIP)-2 and cyclooxygenase (COX)-2 in RAW264.7 cells. Concomitantly, Cd2+ markedly elevated the level of phosphorylated Akt in dose- and time-dependent manners. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked Cd2+-evoked Akt phosphorylation. Correspondingly, LY294002 significantly repressed Cd2+-induced upregulation of MIP-2 and COX-2 in RAW264.7 cells. Further experiments showed that treatment with Cd2+ significantly reduced the level of PTEN protein in RAW264.7 cells. MG132, a specific proteasome inhibitor, blocked Cd2+-induced reduction in PTEN protein as well as Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Of interest, Cd2+-induced degradation of PTEN protein appears to be associated with PTEN ubiquitination. N-acetylcysteine, a glutathione (GSH) precursor, blocked Cd2+-evoked PTEN degradation as well as Akt phosphorylation. By contrast, L-buthionine-S,R-sulfoximine, an inhibitor of cellular GSH synthesis, exacerbated Cd2+-induced PTEN degradation and Akt phosphorylation. Alpha-phenyl-N-tert-butylnitrone and vitamin C, two antioxidants, did not prevent from Cd2+-induced PTEN degradation and Akt phosphorylation. In conclusion, Cd2+ selectively induces MIP-2 and COX-2 through PTEN-mediated PI3K/Akt activation. Cellular GSH depletion mediates Cd2+-induced PTEN degradation and subsequent PI3K/Akt activation in macrophages.
Agglomerated carbon black nanoparticles (CBNPs) administered via respiratory or subcutaneous routes have been shown to promote allergic sensitization to coadministered ovalbumin (OVA) protein in rodents. In the present study, we aimed to model and elucidate the mechanism of this adjuvanticity using an in vitro assay based on T cell sensitization to ovalbumin323–339 peptide (OVAp). CBNP base particles of 22 and 39 nm were characterized and termed CBNP22 and CBNP39 powders. Splenic leukocytes derived from transgenic DO11.10 mice were exposed to suspensions of media alone, concanavalin A mitogen, CBNP agglomerates smaller than 220 nm, OVAp alone, OVAp + anti-CD28 costimulant, OVAp + cyclosporin A immunosuppressant, or OVAp + CBNPs. Samples were analyzed at 72 h post-exposure. Proliferation rate, a marker of cellular mitosis, was assessed. Polymerase chain reaction arrays were used to assess genes involved in allergic response pathways. The mitogen control, costimulatory control, and immunosuppressive control chemicals modified the T helper cell proliferation rate. CBNP22 mildly reduced proliferation at 12 μg/ml, but CBNP39 did not. Gene expression analysis of cells treated with OVAp showed that coincubation with 12 μg/ml CBNP22 enhanced gene expression of interleukin-4 (IL-4), IL-10, and IL-13, all allergy-associated Th2 cytokines. Coincubation of OVAp with 12 μg/ml CBNP39 significantly enhanced IL-13 gene expression concurrent with downregulation of the Th1-associated transcription factor Stat4. IL-4 and IL-13 protein secretion reflected the mRNA trends. The changes were consistently higher in cells exposed to CBNP22 than CBNP39, suggesting that smaller particle size, higher surface area, and higher purity were associated with the direct adjuvant effect on Th2 cells in this genetically susceptible model of OVA allergy.
Vascular leakage is a serious side effect of therapies based on monoclonal antibodies or cytokines which may lead to life-threatening situations. With the steady increase of new drug development programs for large molecules, there is an urgent need for reliable tools to assess this potential liability of new medicines in a rapid and cost-effective manner. Using human umbilical vein endothelial cells (HUVECs) as a model for endothelium, we established an impedance-based assay measuring the integrity of the endothelial cell monolayer in real time. We could demonstrate that the HUVEC monolayer in our system was a relevant model as cells expressed major junctional proteins known to be responsible for maintaining tightness as well as receptors targeted by molecules known to induce vascular leakage in vivo. We assessed the time-dependent loss of barrier function using impedance and confirmed that signals obtained corresponded well to those from standard transwell assays. We assayed a series of reference molecules which led to the expected change of barrier integrity. A nonspecific cytotoxic effect could be excluded by using human fibroblasts as a nonresponder cell line. Finally, we could show reversibility of vascular permeability induced by histamine, IL-1β, or TNF-α by coincubation with established antagonists, further demonstrating relevance of this new model. Taken together, our results suggest that impedance in combination with HUVECs as a specific model can be applied to assess clinically relevant vascular leakage on an in vitro level.
The risk of low birth weights is elevated in prenatal exposure to polycyclic aromatic hydrocarbons (PAHs), which are ubiquitous environmental pollutants generated from combustion of organic compounds, including cigarette smoke. We hypothesized that benzo(a)pyrene (BaP), a member of PAHs existing in cigarette smoke, may affect the myogenesis to cause low birth weights. We investigated the effects of BaP and its main metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), on the myogenic differentiation of human skeletal muscle-derived progenitor cells (HSMPCs). HSMPCs were isolated by a modified preplate technique and cultured in myogenic differentiation media with or without BaP and BPDE (0.25 and 0.5μM) for 4 days. The multinucleated myotube formation was morphologically analyzed by hematoxylin and eosin staining. The expressions of myogenic differentiation markers and related signaling proteins were determined by Western blotting. Both BaP and BPDE at the submicromolar concentrations (0.25 and 0.5μM) dose-dependently repressed HSMPCs myogenic differentiation without obvious cell toxicity. Both BaP and BPDE inhibited the muscle-specific protein expressions (myogenin and myosin heavy chain) and phosphorylation of Akt (a known modulator in myogenesis), which could be significantly reversed by the inhibitors for aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and nuclear factor (NF)-B. BaP- and BPDE-activated NF-B-p65 protein phosphorylation could also be attenuated by both AhR and ER inhibitors. The inhibitory effects of BaP and BPDE on myogenesis were reversed after withdrawing BaP exposure, but not after BPDE withdrawal. These results suggest that both BaP and BPDE are capable of inhibiting myogenesis via an AhR- or/and ER-regulated NF-B/Akt signaling pathway.
As nanoparticles could form aggregates in biological systems, the dynamics of their dispersity drives the temporal effect of nanoparticles in vitro. To test this hypothesis, the fumed silica nanoparticles (SiNPs) that have primary sizes of 7–14 nm and form aggregates in culture medium were selected for toxicity study in human lung A549 cells. The dispersity of SiNPs was analyzed by dynamic light scattering and transmission of electron microscopy. Cytotoxicity assays including mitochondrial activity, intracellular level of reactive oxygen species (ROS), and membrane damage together with the 1H-NMR-based extracellular metabonomic assay were conducted to determine the temporal dose-effect relationship of SiNPs. In cell culture medium, SiNPs dispersed well initially at 25–100 μg/ml; however, they sedimented rapidly in a concentration-dependent manner. SiNPs caused a dose-dependent increase of intracellular ROS and cell membrane damage at 4 h and a loss of cell viability after 48 h. SiNPs also induced an elevation of extracellular glucose, lactate, phenylalanine, histidine, and tyrosine levels in a time- and concentration-dependent manner. The dose-effect patterns at 4 h were different from that at 12 and 24 h as assessed by both cytotoxicity and metabonomic assays. Both fitted better with polynomial regression than linear regression, implying multimode action of SiNPs at different concentrations. The early NP-cell interaction and the late sedimentation could be attributable to the temporal effects of SiNPs. The extracellular 1H-NMR-based metabonomics demonstrated a potential as a robust nondestructive tool for monitoring the temporal effect of NPs that tend to aggregate in nature.
The potential uses of engineered C60 fullerene (C60) have expanded in recent decades to include industrial and biomedical applications. Based on clinical findings associated with particulate matter exposure and our data with multi-walled carbon nanotubes, we hypothesized that ischemia/reperfusion (I/R) injury and pharmacological responses in isolated coronary arteries would depend upon the route of exposure and gender in rats instilled with C60. Male and female Sprague Dawley rats were used to test this hypothesis by surgical induction of cardiac I/R injury in situ 24 h after intratracheal (IT) or intravenous (IV) instillation of 28 μg of C60 formulated in polyvinylpyrrolidone (PVP) or PVP vehicle. Serum was collected for quantification of various cytokines. Coronary artery segments were isolated for assessment of vasoactive pharmacology via wire myography. Both IV and IT exposure to C60 resulted in expansion of myocardial infarction in male and female rats following I/R injury. Serum-collected post-I/R showed elevated concentrations of interleukin-6 and monocyte chemotactic protein-1 in male rats exposed to IV C60. Coronary arteries isolated from male rats exposed to IT C60 demonstrated augmented vasocontraction in response to endothelin-1 that was attenuated with Indomethacin. IV C60 exposure resulted in impaired acetylcholine relaxation in male rats and IT C60 exposure resulted in depressed vasorelaxation in response to sodium nitroprusside in female rats. Based on these data, we conclude that IT and IV exposure to C60 results in unique cardiovascular consequences that may favor heightened coronary resistance and myocardial susceptibility to I/R injury.
We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca2+-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (–)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (–)-PCB 136 is observed at concentrations ranging from 0.1 to 100nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca2+-sensitive dye demonstrates that (–)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca2+ oscillations. Similarly, (–)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.
Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20–22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.
2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) has been associated with many disease states in humans. A rising concern is that exposure early in life can lead to adult toxicity and toxicity in subsequent generations. Juvenile zebrafish exposed to TCDD (50 pg/ml in water; 1 h exposure) at 3 and 7 weeks post fertilization showed toxicity only later in adulthood. We have maintained the offspring of these exposed F0 fish to determine whether we could find adverse affects in the next two generations of F1 and F2 offspring. TCDD exposure produced a significantly higher female:male ratio in all three generations. Scoliosis-like axial skeleton abnormalities, not normally observed in controls, were present in the F1 and F2 generations descended from the treated F0 founders. Egg release and fertilization success were reduced in the TCDD lineage F1 and F2 generations. This reduction in fertility in the TCDD lineage F2 generation could be attributed to alterations in the F2 males. Using zebrafish as a model allowed the simultaneous maintenance of different generations with relatively small space and costs. The zebrafish showed clear signs of transgenerational responses persisting into generations never directly exposed to TCDD.
Formaldehyde is a nasal carcinogen in rodents at high doses and is an endogenous compound that is present in all living cells. Due to its high solubility and reactivity, quantitative risk estimates for inhaled formaldehyde have relied on internal dose estimates in the upper respiratory tract. Dosimetry calculations are complicated by the presence of endogenous formaldehyde concentrations in the respiratory mucosa. Anatomically accurate computational fluid dynamics (CFD) models of the rat, monkey, and human nasal passages were used to simulate uptake of inhaled formaldehyde. An epithelial structure was implemented in the nasal CFD models to estimate formaldehyde absorption from air:tissue partitioning, species-specific metabolism, first-order clearance, DNA binding, and endogenous formaldehyde production. At an exposure concentration of 1 ppm, predicted formaldehyde nasal uptake was 99.4, 86.5, and 85.3% in the rat, monkey, and human, respectively. Endogenous formaldehyde in nasal tissues did not significantly affect wall mass flux or nasal uptake predictions at exposure concentrations > 500 ppb; however, reduced nasal uptake was predicted at lower exposure concentrations. At an exposure concentration of 1 ppb, predicted nasal uptake was 17.5 and 42.8% in the rat and monkey; net desorption of formaldehyde was predicted in the human model. The nonlinear behavior of formaldehyde nasal absorption will affect the dose-response analysis and subsequent risk estimates at low exposure concentrations. Updated surface area partitioning of nonsquamous epithelium and average flux values in regions where DNA-protein cross-links and cell proliferation rates were measured in rats and monkeys are reported for use in formaldehyde risk models of carcinogenesis.
Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints below, at (~75 mg/kg-day), and above the carcinogenic dose level of PB and examine these responses using benchmark dose modeling. Linear toxicokinetics were observed for all doses. Increased liver weight, hepatocellular hypertrophy, and mitotic figures were seen at 75 and 150 mg/kg-day. CAR activation, based on Cyp2b qPCR and pentoxyresorufin dealkylase activity, occurred at doses ≥ 1.5 mg/kg-day. The no-observable transcriptional effect level for global gene expression was 15 mg/kg-day. At 2 days, several xenobiotic metabolism and cell protective pathways were activated at lower doses and to a greater degree in females. However, hepatocellular proliferation, quantified by bromodeoxyuridine immunohistochemistry, was the most sensitive indicator of PB exposure with female mice more sensitive than males, contrary to sex-specific differences in sensitivity to hepatocarcinogenesis. Taken together, the identification of low-dose cellular and molecular transitions in the subtumorigenic dose range aids the understanding of early key events in CAR-mediated hepatocarcinogenesis.
Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in time- and concentration-dependent manners (0.86 g ± 0.11 at 10μM simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca2+ channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca2+ increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonist-induced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca2+ influx and RhoA activity, ultimately leading to apoptosis and structural remodeling.
Ipratropium bromide, a nonselective muscarinic antagonist, is widely prescribed for the treatment of chronic obstructive pulmonary disease (COPD). Analyses of COPD patients, with underlying ischaemic heart disease, receiving anticholinergics, have indicated increased risk of severity and occurrence of cardiovascular events (including myocardial infarction). The present study explored whether ipratropium bromide induces myocardial injury in nonclinical models of simulated myocardial ischaemia/reperfusion injury. Adult Sprague Dawley rat hearts/primary ventricular myocytes were exposed to simulated ischaemia/hypoxia prior to administration of ipratropium at the onset of reperfusion/reoxygenation. Infarct to risk ratio and cell viability was measured via triphenyl tetrazolium chloride staining and thiazolyl blue tetrazolium bromide (MTT) assay. The involvement of apoptosis and necrosis was evaluated by flow cytometry. Mitochondrial-associated responses were detected by tetramethylrhodamine methyl ester fluorescence and myocyte contracture. Ipratropium (1 x 10–11 M – 1 x 10–4 M) significantly increased infarct/risk ratio and decreased cell viability in a dose-dependent manner. Increased levels of necrosis and apoptosis were observed via flow cytometry, accompanied by increased levels of cleaved caspase-3 following ipratropium treatment. Levels of endogenous myocardial acetylcholine were verified via use of an acetylcholine assay. In these experimental models, exogenous acetylcholine (1 x 10–7 M) showed protective properties, when administered alone, as well as abrogating the exacerbation of myocardial injury during ischaemia/reperfusion following ipratropium coadministration. In parallel experiments, under conditions of myocardial ischaemia/reperfusion, a similar injury was observed following atropine (1 x 10–7 M) administration. These data demonstrate for the first time in a nonclinical setting that ipratropium exacerbates ischaemia/reperfusion injury via apoptotic- and necrotic-associated pathways.
This study aims to test the hypothesis that thiazolidinedione rosiglitazone (RSG), a selective peroxisome proliferator-activated receptor (PPAR) agonist, causes cardiotoxicity independently of PPAR. Energy metabolism and mitochondrial function were measured in perfused hearts isolated from C57BL/6, cardiomyocyte-specific PPAR-deficient mice, and their littermates. Cardiac function and mitochondrial oxidative stress were measured in both in vitro and in vivo settings. Treatment of isolated hearts with RSG at the supratherapeutic concentrations of 10 and 30μM caused myocardial energy deficiency as evidenced by the decreases in [PCr], [ATP], ATP/ADP ratio, energy charge with a concomitant cardiac dysfunction as indicated by the decreases in left ventricular systolic pressure, rates of tension development and relaxation, and by an increase in end-diastolic pressure. When incubated with tissue homogenate or isolated mitochondria at these same concentrations, RSG caused mitochondrial dysfunction as evidenced by the decreases in respiration rate, substrate oxidation rates, and activities of complexes I and IV. RSG also increased complexes I- and III-dependent O2– production, decreased glutathione content, inhibited superoxide dismutase, and increased the levels of malondialdehyde, protein carbonyl, and 8-hydroxy-2-deoxyguanosine in mitochondria, consistent with oxidative stress. N-acetyl-L-cysteine (NAC) 20mM prevented RSG-induced above toxicity at those in vitro settings. Cardiomyocyte-specific PPAR deletion and PPAR antagonist GW9662 did not prevent the observed cardiotoxicity. Intravenous injection of 10 mg/kg RSG also caused cardiac dysfunction and oxidative stress, 600 mg/kg NAC antagonized these adverse effects. In conclusion, this study demonstrates that RSG at supratherapeutic concentrations causes cardiotoxicity via a PPAR-independent mechanism involving oxidative stress-induced mitochondrial dysfunction in mouse hearts.