Publications (142) View all
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Article: Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.
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ABSTRACT: Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5mmol/kg/day via drinking water for 30days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.Toxicology and Applied Pharmacology 01/2013; · 4.45 Impact Factor -
Article: Hepatic lipid profiling of deer mice fed ethanol using (1)H and (31)P NMR spectroscopy: A dose-dependent subchronic study.
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ABSTRACT: Chronic alcohol abuse is a 2nd major cause of liver disease resulting in significant morbidity and mortality. Alcoholic liver disease (ALD) is characterized by a wide spectrum of pathologies starting from fat accumulation (steatosis) in early reversible stage to inflammation with or without fibrosis and cirrhosis in later irreversible stages. Previously, we reported significant steatosis in the livers of hepatic alcohol dehydrogenase (ADH)-deficient (ADH(-)) vs. hepatic ADH-normal (ADH(+)) deer mice fed 4% ethanol daily for 2months [Bhopale et al., 2006, Alcohol 39, 179-188]. However, ADH(-) deer mice fed 4% ethanol also showed a significant mortality. Therefore, a dose-dependent study was conducted to understand the mechanism and identify lipid(s) involved in the development of ethanol-induced fatty liver. ADH(-) and ADH(+) deer mice fed 1, 2 or 3.5% ethanol daily for 2months and fatty infiltration in the livers were evaluated by histology and by measuring dry weights of extracted lipids. Lipid metabolomic changes in extracted lipids were determined by proton ((1)H) and (31)phosphorus ((31)P) nuclear magnetic resonance (NMR) spectroscopy. The NMR data was analyzed by hierarchical clustering (HC) and principle component analysis (PCA) for pattern recognition. Extensive vacuolization by histology and significantly increased dry weights of total lipids found only in the livers of ADH(-) deer mice fed 3.5% ethanol vs. pair-fed controls suggest a dose-dependent formation of fatty liver in ADH(-) deer mouse model. Analysis of NMR data of ADH(-) deer mice fed 3.5% ethanol vs. pair-fed controls shows increases for total cholesterol, esterified cholesterol, fatty acid methyl esters (FAMEs), triacylglycerides and unsaturation, and decreases for free cholesterol, phospholipids and allylic and diallylic protons. Certain classes of neutral lipids (cholesterol esters, fatty acyl chain (COCH(2)) and FAMEs) were also mildly increased in ADH(-) deer mice fed 1 or 2% ethanol. Only small increases were observed for allylic and diallylic protons, FAMEs and unsaturations in ADH(+) deer mice fed 3.5% ethanol vs. pair-fed controls. PCA of NMR data showed increased clustering by gradual separation of ethanol-fed ADH(-) deer mice groups from their respective pair-fed control groups and corresponding ethanol-fed ADH(+) deer mice groups. Our data indicate that dose of ethanol and hepatic ADH deficiency are two key factors involved in initiation and progression of alcoholic fatty liver disease. Further studies on characterization of individual lipid entities and associated metabolic pathways altered in our deer mouse model after different durations of ethanol feeding could be important to delineate mechanism(s) and identify potential biomarker candidate(s) of early stage ALD.Toxicology and Applied Pharmacology 08/2012; 264(3):361-9. · 4.45 Impact Factor -
Article: Rickettsiae induce microvascular hyperpermeability via phosphorylation of VE-cadherins: evidence from atomic force microscopy and biochemical studies.
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ABSTRACT: The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein-protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses.PLoS Neglected Tropical Diseases 06/2012; 6(6):e1699. · 4.69 Impact Factor -
Article: Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy.
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ABSTRACT: Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation ((3)H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80 μM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140 μM) markedly decreased MTT absorbance (40%) at 120 h. VR or VS treatment inhibited (3)H-thymidine incorporation (24h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear "vacuoles"). TEM revealed perinuclear "vacuoles" to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP(+) puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.Toxicology and Applied Pharmacology 05/2012; 262(3):265-72. · 4.45 Impact Factor -
Article: Spontaneous occurrence of dissecting aneurysms in the region of the ductus arteriosus in four-day-old Wistar rat pups.
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ABSTRACT: Dissecting aortic aneurysms, generally involving the thoracic aorta, have been shown to be caused by specific aliphatic amines in developing rats. Whether such lesions might occur spontaneously in control rats is not known. Therefore, in this study, 1,016 four-day-old, untreated rats culled from ongoing scheduled breeding studies were subjected to gross and histopathological examination in order to create a background control data base on the incidence of spontaneous aortic dissecting aneurysms. Two animals (0.2%) were found to have small dissecting aortic aneurysms, and an additional 2 (0.2%) had only hemorrhagic lesions. All of these lesions were limited to the region of the ductus arteriosus. An additional 18 findings were judged to be artifacts. These findings suggest that small vascular dissections may rarely occur in the aortic arch adjacent to the ductus arteriosus. Special attention should be paid in experimental studies to avoid confusing these small spontaneous lesions with treatment-induced lesions or artifacts.Toxicologic Pathology 10/2011; 39(6):969-74. · 1.91 Impact Factor
