Dimethyl Sulfoxide Control Research Articles

S113 Haemophilus influenzae stimulation of alveolar macrophages from COPD patients; effects of corticosteroids

BackgroundThe lower airways of COPD patients are often chronically colonised by bacteria such as Non-typeable Haemophilus influenza (NTHI). Bacteria are a common cause of COPD exacerbations. Corticosteroids are often used...

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Selective cyclooxygenase-1 inhibition improves collateral vascular reactivity in biliary cirrhotic rats

BackgroundEvidence has demonstrated that overproduction of prostacyclin (PGI2) is critical in the pathogenesis of splanchnic hyposensitivity to vasoconstrictors in the cirrhotic state. The biosynthesis of PGI2 is through cyclooxygenase (COX). This study evaluated which isoform of COX is dominant in the mechanism of collateral vascular reactivity of biliary cirrhotic rats. MethodsThree groups of formalin-injected common bile duct-ligated (FBDL) induced cirrhotic rats received two doses of: (1) selective COX-1 inhibitor (SC-560 2 mg/kg); (2) COX-2 inhibitor (NS-398 2 mg/kg); (3) dimethyl sulfoxide (control). Subsequently, the rats were kept in metabolic cages for 24 hours to collect urine. Thereafter, the systemic and portal hemodynamics and renal function were measured. In another series, using in-situ collateral perfusion model, the collateral vascular responses to arginine vasopressin (AVP) were measured in the subject rats after preincubation of vehicle (Krebs solution), SC-560 (5 μM) or NS-398 (10 μM). ResultsThe mean arterial pressure, heart rate, and portal pressure were similar among SC-560-treated, NS-398-treated, and control groups. Additionally, there was no significant difference in the calculated creatinine clearance rates among these three groups. SC-560 preincubation significantly enhanced the pressor effect of AVP at the concentration of 3M × 10−9 M (11.0 ± 1.0 mmHg vs. 6.4 ± 0.6 mmHg, p = 0.002) in the cirrhotic rats. ConclusionThere was no significant hemodynamic change and renal toxicity after acute administration of COX inhibitor in the FBDL-induced cirrhotic rats. Preincubation of selective COX-1, but not COX-2, inhibitor could enhance collateral vascular response to AVP, indicating that COX-1 plays a major role in the collateral vascular reactivity.

Characterization of Reference Genes in Rare Minnow, <I>Gobiocypris Rarus</I> (Actinopterygii: Cypriniformes: Cyprinidae), in Early Postembryonic Development and in Response to Edcs Treatment

Background. Endocrine disrupting chemicals (EDCs) are natural and anthropogenic compounds discharged into the environment known to disrupt the endocrine system of humans and animals by mimicking functions of steroids in vivo. Many important events occurring during early postembryonic development, in relation to the gene expression attracted our attention. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible method for gene expression analysis, with gene expression levels quantified by normalization to reference gene. The aim of this study was to select the suitable reference gene after EDCs exposure and during early postembryonic development. Materials and methods. For the study of the fish age effect, juveniles of Gobiocypris rarus Ye et Fu, 1983, were obtained at: 18, 22, 26, 30, 34, 38, 42, 46, and 50 days post fertilization (dpf). For mRNA expression analysis of the juvenile fish after EDCs treatment, the juveniles at 31 dpf were exposed to bisphenol A (BPA) (10 nM) and 17α-ethinylestradiol (EE2) (1 nM), respectively dissolved in dimethyl sulfoxide (DMSO) or solvent (0.001% DMSO, v/v) control group for 3 days. Cq values of the reference genes were obtained using qRT-PCR. The stability of these reference genes was analyzed by BestKeeper, geNorm, and NormFinder software, respectively. The expression of each reference gene was calculated using the 2–ΔCq method. In parallel, the mRNA expressions of cyp19a1b were normalized by the single most/least stable reference gene and the combinations of top-ranked reference genes.  Results. In this study, six candidate reference genes, actb, ef1a, gapdh, g6pd, tbp, and tuba1, were chosen to analyze their expression stability in relation to fish age and in the juvenile fish exposed to BPA and EE2. During early postembryonic development of Gobiocypris rarus, actb，ef1a, and gapdh were identified as the most stably expressed reference genes. In the juvenile fish exposed to BPA and EE2 for three days, gapdh, and actb were the most stable. However, g6pd and tuba1 were identified as the least stably expressed genes during the early postembryonic development and under BPA and EE2 exposure.  Conclusion. The presently reported study suggested that the mRNA expressions of the reference genes could be affected by chemical exposure or different physiological periods. In addition, it was indicated that stable reference gene should be selected to normalize the target gene expression to assure the correctness and accuracy of the experiment results. The last but not the least, we successfully obtained five commonly used reference genes of Gobiocypris rarus Ye et Fu, 1983, which can be applied in future studies serving as the stable reference gene and providing a broader range of selecting the stable reference gene.

Short-Course Rapamycin Treatment Preserves Airway Epithelium and Protects Against Bronchiolitis Obliterans

Damage to airway epithelium is closely related to the development of bronchiolitis obliterans (BO) in pulmonary transplantation. Rapamycin protects against BO development in a murine model, but its use in patients undergoing lung transplantation is limited by its side effects. We hypothesized that short-course rapamycin dosing could be used to prevent airway epithelium loss and protect against BO development in a murine model. A total alloantigenic mismatch, murine, heterotopic tracheal transplant model of BO was used. Animals were treated with either rapamycin or dimethyl sulfoxide (controls) according to one of three treatment regimens: (1) days 1 through 14 after transplantation, (2) days 3 through 7 after transplantation, or (3) days 14 through 28 after transplantation. Epithelial loss was assessed by use of hematoxylin and eosin stains 14 and 28 days after transplantation. Tracheal luminal obliteration was assessed at 28 days. Early rapamycin treatment was protective against epithelial loss 14 days after transplantation in comparison with control animals (p < 0.001). Rapamycin treatment from days 1 to 14 was more effective at epithelial preservation (p = 0.002) and reducing luminal obliteration (p < 0.001) at 28 days than was rapamycin treatment from days 3 to 7. Late rapamycin treatment (days 14 to 28) allowed for recovery of the previously denuded epithelium at 28 days (92.5% epithelial loss to 35.6%) and a reduction in BO (p < 0.001). Short-course rapamycin treatment protects against airway epithelium loss and subsequent development of BO in a murine model. Because of its immunosuppressive and antifibrotic effects, rapamycin may prove to be the ideal medication to prevent chronic rejection and BO in patients undergoing lung transplantation.

Ubiquinol rescues simvastatin-suppression of mitochondrial content, function and metabolism: Implications for statin-induced rhabdomyolysis

Statin medications diminish cholesterol biosynthesis and are commonly prescribed to reduce cardiovascular disease. Statins also reduce production of ubiquinol, a vital component of mitochondrial energy production; ubiquinol reduction may contribute to rhabdomyolysis. Human rhabdomyosarcoma cells were treated with either ethanol and dimethyl sulfoxide (DMSO) control, or simvastatin at 5µM or 10µM, or simvastatin at 5µM with ubiquinol at 0.5µM or 1.0µM for 24h or 48h. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunocytochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. Treatment of human rhabdomyosarcoma cells with simvastatin significantly reduced oxidative, total metabolism, and cellular ATP content in a time- and dose-dependent manner which was rescued by concurrent treatment with ubiquinol. Treatment with simvastatin significantly reduced mitochondrial content as well as cell viability which were both rescued by simultaneous treatment with ubiquinol. This work demonstrates that the addition of ubiquinol to current statin treatment regimens may protect muscle cells from myopathies.

A three‐generational study of In ovo exposure to PBDE‐99 in the zebra finch

Based on a literature review of avian data for polybrominated diphenyl ethers (PBDEs), ecologically relevant doses, low (10 ng/egg), medium (100 ng/egg), and high (1,000 ng/egg) of the 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) congener along with dimethylsulfoxide (DMSO) control were injected into the yolk sac of un-incubated eggs of zebra finch, Taeniopygia guttata. Offspring development and adult phenotype were followed over three generations. No effects of in ovo PBDE exposure on hatching success, chick growth, thyroid hormone levels, or hematological traits were measured at sexual maturity (90 d posthatching). However, the authors did detect significant effects of BDE-99 treatment on adult phenotype of in ovo-exposed birds by breeding observations, in which clutch size was significantly smaller in all PBDE-dosed birds (low, medium, and high) compared with controls. A trend was also seen for longer laying intervals in PBDE-dosed birds (13-14 d) compared with control birds (8 d). In addition, a significant effect of PBDE was found on growth of the second-generation offspring of in ovo-treated females; body mass was significantly lower in the high-PBDE dosed birds compared with controls from hatch through to fledging (day 30). The authors found no evidence of effects over the longer term and in successive generations, whether in adult, reproductive phenotype of the second-generation offspring of in ovo-treated birds, or in the growth of their (third-generation) offspring. Their results suggest that egg levels as low as 10 ng/g BDE-99 may affect reproduction in small passerines by reducing clutch size.

Bisphenol A and Expression of Cell Cycle Regulators and Apoptotic Factors in Cultured Mouse Ovarian Antral Follicles.

Bisphenol A (BPA) is a plasticizer that is widely used in polycarbonate plastics. BPA is a known endocrine disrupting chemical that has been reported to have some toxic effects on female reproductive system. For example, BPA exposure has been inversely associated with the number and quality of oocytes retrieved from women undergoing in-vitro fertilization. Our previous studies indicated that a 100 μg/ml dose of BPA inhibits mouse antral follicle growth in culture. However, the effects of lower dose BPA exposure on mouse antral follicles are not known. Thus, we hypothesized that low doses of BPA (0.001-1 μg/ml) inhibit follicle growth by altering expression levels of key cell cycle regulators and/or apoptotic factors. To test this hypothesis, we mechanically isolated antral follicles from mouse ovaries (C57Bl/6; aged 50-54 days; n=3) and cultured them in dimethylsulfoxide (DMSO) or BPA (0.001-100 μg/ml) for 96 hours. Every 24 hours, follicle growth was measured in perpendicular axes. At the end of the cultures, follicles were snap-frozen and further processed for real-time polymerase chain reactions for expression levels of the following genes: transformation related protein (Trp53), B-cell leukemia/lymphoma 2 (Bcl2), Bcl-2–associated X protein (Bax), Bcl-2-associated agonist of cell death (Bad), cyclin-D2 (Ccnd2), Cyclin-dependent kinase 4 (Cdk4), and cyclin-E1 (Ccne1). The data indicate that low doses of BPA (0.001-1μg/ml) do not inhibit follicle growth, but that the higher dose of BPA (100 μg/ml) significantly inhibits follicle growth compared to DMSO control. Further, low doses of BPA did not alter expression levels of the selected genes compared to DMSO control. These data suggest that treatment of mouse antral follicles with relatively low doses of BPA does not affect the growth or levels of selected cell cycle regulators or apoptotic factors in mouse antral follicles. Supported by: NIH ES019178 (JAF) and Billie Field Fellowships (ZRC and WW).

DMSO exhibits similar cytotoxicity effects to thalidomide in mouse breast cancer cells

The purpose of this study was to evaluate the cytotoxic effect of thalidomide on 4T1 and 4THMpc mouse breast cancer cell lines. Mouse breast cancer cells (4T1) and cells derived from metastatic lesions (4THMpc) were treated with various doses of thalidomide [10(-2)-100 µM dissolved in dimethyl sulfoxide (DMSO) as recommended] and 1.4 µM DMSO (maximum DMSO concentration in the highest thalidomide dose) as a DMSO control against the untreated control groups. MTT was used to evaluate the cytotoxic effects of the treatments. Therefore, we investigated the role of thalidomide on apoptosis. A fluorometric EnzChek caspase-3 enzyme activity assay kit was used to evaluate the apoptotic effects of thalidomide. Thalidomide dissolved in DMSO exhibited cytotoxic effects on 4T1 and 4THMpc cells compared to the control groups incubated without any supplement. Treatment with thalidomide resulted in apoptosis of mouse breast cancer cells in a time- and dose-dependent manner as demonstrated by caspase-3 enzyme activity. However, DMSO alone suppressed cell proliferation more effectively than thalidomide. In cultured mouse breast cancer cells the inhibitory effect of thalidomide may be partially attributed to the solvent DMSO alone.

Pooled safety analysis of diclofenac sodium topical solution 1.5% (w/w) in the treatment of osteoarthritis in patients aged 75 years or older

BackgroundThis study aimed to determine the safety of diclofenac sodium topical solution 1.5% (w/w) in 45.5% dimethyl sulfoxide (TDiclo) for the treatment of knee or hand osteoarthritis in persons aged 75 years or older.MethodsA pooled analysis of safety data from seven multicenter, randomized, blinded, Phase III clinical trials (4–12 weeks’ duration) of TDiclo was conducted. The analysis focused on a subset of patients (n = 280) aged 75 years or older with a primary diagnosis of osteoarthritis of the knee (six trials) or hand (one trial). Patients received one of three topical treatments: TDiclo (n = 138); placebo (2.33% or 4.55% dimethyl sulfoxide, n = 39); or control (45.5% dimethyl sulfoxide, n = 103). Treatment groups were compared using Chi-square analysis, Fisher’s Exact test, or analysis of variance.ResultsThe most common adverse events involved the skin or subcutaneous tissue, primarily at the application site. The incidence of dry skin was higher in the TDiclo (36.2%; P < 0.0001) and dimethyl sulfoxide control (18.4%; P = 0.0142) groups than in the placebo group (2.6%); the incidence of other skin or subcutaneous tissue adverse events was similar between the groups. Relatively few patients (<18%) experienced gastrointestinal adverse events, and group differences were not detected. In the TDiclo group, constipation (3.6%), diarrhea (3.6%), and nausea (3.6%) were the most common gastrointestinal adverse events. Cardiovascular and renal/ urinary adverse events were rare, and group differences were not detected. There was one case (0.7%) each of hypertension, spider veins, and vasodilation in the TDiclo group. Changes from baseline to the final visit in blood pressure and hepatic/renal enzyme levels were also similar between the groups.ConclusionTDiclo appears to be well tolerated for the treatment of osteoarthritis in persons aged 75 years or older.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors invitro and their recovery in transplanted recipients invivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Characterization of Novel Synthesized Small Molecular Compounds Against Non-Small Cell Lung Cancer

With the exception of surgery, the standard platinum-based chemotherapeutic agents are the preferred treatment for non-small cell lung cancer (NSCLC); however little improvement (5-year survival) has been made. Therefore it is highly desirable to develop innovative therapeutic agents for NSCLC treatment. Highly enantioselective synthetic methods were developed and a broad compound library was established. Cell toxicity, cell sensitivity, cell proliferation, cell invasion, and three-dimensional colony formation assays were used to assess the anticancer potential of these compounds in non-small-cell lung cancer (NSCLC) cell lines. We found that the S-form of compound PL54 (PL54S, 5-20 μM) exhibited strong anticancer activity in 5 tested NSCLC cell lines. We further synthesized a highly pure R-form enantiomer of PL54 (PL54R) and its racemate (PL54Rac) and characterized their anticancer activities. The results showed that PL54S is more potent than PL54R and PL54Rac against the tested cell lines. Furthermore, less cellular toxicity was observed in the normal human lung fibroblasts. Similarly, PL54S displayed greater anti-colony formation activity compared with PL54R and PL54Rac. The cellular sensitivity assay revealed that PL54S and PL54Rac significantly suppressed cologenic formation compared with PL54R and dimethyl sulfoxide controls (p<0.01). All PL54 compounds (5 to 20 μM) significantly inhibited cell proliferation and invasion of the A549 cell line (p<0.01). A soft agar colony formation assay revealed that PL54S and PL54Rac (10 mM), but not PL54R, significantly inhibited colony formation of tested NSCLC cells (p<0.01). The stereospecific compounds may prove to be a novel technique for the treatment of NSCLC.

Controls on Sr/Ca and Mg/Ca in scleractinian corals: The effects of Ca-ATPase and transcellular Ca channels on skeletal chemistry

The Sr/Ca of aragonitic coral skeletons is a commonly used palaeothermometer. However skeletal Sr/Ca is typically dominated by weekly–monthly oscillations which do not reflect temperature or seawater composition and the origins of which are currently unknown. To test the impact of transcellular Ca2+ transport processes on skeletal Sr/Ca, colonies of the branching coral, Pocillopora damicornis, were cultured in the presence of inhibitors of Ca-ATPase (ruthenium red) and Ca channels (verapamil hydrochloride). The photosynthesis, respiration and calcification rates of the colonies were monitored throughout the experiment. The skeleton deposited in the presence of the inhibitors was identified (by 42Ca spike) and analysed for Sr/Ca and Mg/Ca by secondary ion mass spectrometry. The Sr/Ca of the aragonite deposited in the presence of either of the inhibitors was not significantly different from that of the solvent (dimethyl sulfoxide) control, although the coral calcification rate was reduced by up to 66% and 73% in the ruthenium red and verapamil treatments, respectively. The typical precision (95% confidence limits) of mean Sr/Ca determinations within any treatment was <±1% and differences in skeletal Sr/Ca between treatments were correspondingly small. Either Ca-ATPase and Ca channels transport Sr2+ and Ca2+ in virtually the same ratio in which they are present in seawater or transcellular processes contribute little Ca2+ to the skeleton and most Ca is derived from seawater transported directly to the calcification site. Variations in the activities of Ca-ATPase and Ca-channels are not responsible for the weekly–monthly Sr/Ca oscillations observed in skeletal chronologies, assuming that the specificities of Ca transcellular transport processes are similar between coral genera.

Notch1 Regulates the Effects of Matrix Metalloproteinase-9 on Colitis-Associated Cancer in Mice

Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9. Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA. MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Evaluation of the Toxicity of Triamcinolone Acetonide and Dexamethasone Sodium Phosphate on Human Lens Epithelial Cells (HLE B-3)

The purpose of this study was to compare the in vitro effects of triamcinolone acetonide (TA) and dexamethasone sodium phosphate (DEX) on human lens epithelial cells (HLE B-3). HLE B-3 cells were exposed for 24 h to commercially available TA (c-TA) and dimethylsulfoxide-solubilized TA (s-TA). The cells were treated with 1,000 (clinical dose), 750, 500, 200, and 100 μg/mL concentrations of c-TA, s-TA, and supernatant for 24 h. The cells were also treated with DEX at 2, 1, 0.5, 0.2, 0.1 (clinical dose), and 0.05 mg/mL. Cell viability, caspase-3/7 activity, and DNA fragmentation analyses were performed. The mean cell viabilities of HLE B-3 after exposure to c-TA at 1,000, 750, 500, 200, and 100 μg/mL were significantly reduced compared with control untreated cells. The s-TA also significantly reduced cell viability at 1,000, 750, and 500 μg/mL compared with dimethylsulfoxide control. The supernatant did not reduce cell viability. Caspase-3/7 activity significantly increased after treatment with c-TA and s-TA. DNA laddering revealed bands at 200 bp intervals with both c-TA at≥100 μg/mL and s-TA at ≥500 μg/mL. The cell viabilities of HLE B-3 after 24 h exposure to DEX were significantly reduced at 2 and 1 mg/mL but not at lower concentrations tested. Caspase-3/7 activities in HLE B-3 cells were not increased significantly after treatment with DEX at any dose tested. DNA laddering did not reveal any band at any dose tested. This study showed that TA at its clinical dose (1,000 μg/mL) in both commercial preparation and solubilized forms decrease HLE B-3 cell viability through an apoptotic pathway. DEX at its clinical dose (0.1 mg/mL) does not decrease cell viability or cause any increase of caspase-3/7 activity. This study suggests that for long-term sustained-release devices, DEX may be less damaging to human lens cells than TA.

Micronucleus frequency in sheep lymphocytes after in vitro exposure to fungicide tolylfluanid

The fungicide tolylfluanid (N - dichlorofluoromethylthio-N′, N - dimethyl - N - p - tolylsulfamide), was investigated by cytokinesis-block micronucleus assay. Tolylfluanid at the lowest concentration (1 × 10− 6mol L− 1)did not influence significantly the frequency of micronuclei in sheep lymphocyte cultures in comparison with control (32.33 ± 3.51/1000 binucleated cells versus 30.33 ± 2.82/1000 binucleated cells in dimethylsulfoxide (DMSO) control, P = 0.44). Higher tolylfluanid concentrations (1 × 10− 4 and, 1 × 10− 5 mol L− 1) resulted in a significant dose-dependent increase in the number of micronuclei in comparison with control (74.00 ± 13.00/1000 binucleated cells and 52.67 ± 10.12/1000 binucleated cells versus 30.33 ± 2. 82/1000 binucleated cells in DMSO control, P = 0.005 and 0.02, respectively, ANOVA followed by Tukey test P < 0.05). Many of the treated cells also possessed multiple micronuclei. Tolylfluanid did not affect the nuclear division index at all treatment concentrations. Our in vitro results thus demonstrate that tolylfluanid had a significant genotoxic effect at only the highest concentration tested.

Expression of CYP1C1 and CYP1A in Fundulus heteroclitus during PAH-induced carcinogenesis

CYP1C1 is a relatively newly identified member of the cytochrome P450 family 1 in teleost fish. However, CYP1C1's expression and physiological roles relative to the more recognized CYP1A in polycyclic aromatic hydrocarbons (PAHs) induced toxicities are unclear. Fundulus heteroclitus fry were exposed at 6–8 days post-hatch (dph) and again at 13–15 dph for 6 h to dimethyl sulfoxide (DMSO) control, 5 mg/L benzo[ a]pyrene (BaP), or 5 mg/L dimethylbenzanthracene (DMBA). Fry were euthanized at 0, 6, 18, 24 and 30 h after the second exposure. In these groups, both CYP1A and CYP1C1 protein expression were induced within 6 h after the second exposure. Immunohistochemistry (IHC) results from fry revealed strongest CYP1C1 expression in renal tubular and intestinal epithelial cells. Additional fish were examined for liver lesions 8 months after initial exposure. Gross lesions were observed in 20% of the BaP and 35% of the DMBA-treated fish livers. Histopathologic findings included foci of cellular alteration and neoplasms, including hepatocellular adenoma, hepatocellular carcinoma and cholangioma. Strong CYP1A immunostaining was detected diffusely in altered cell foci and on the invading margin of hepatocelluar carcinomas. Lower CYP1A expression was seen in central regions of the neoplasms. In contrast, CYP1C1 was only detectable and highly expressed in proliferated bile duct epithelial cells. Our CYP1C1 results suggest the potential for tissue specific CYP1C1-mediated PAH metabolism but not a more chronic role in progression to liver hepatocellular carcinoma.

Neurotensin modulates central muscarinic receptors, an effect which does not involve the high-affinity neurotensin receptor (NTS1)

Neurotensin (NT) is a tridecapeptide distributed in central and peripheral nervous systems, which can behave as a neurotransmitter or neuromodulator at central and peripheral levels. Herein we tested the potential effect of this peptide on quinuclidinyl benzilate ([ 3H]-QNB) binding to muscarinic receptor in rat CNS membranes. It was observed that NT decreased up to 50–70% ligand binding at 1 × 10 − 7 M–1 × 10 − 5 M concentration in cerebral cortex, cerebellum and striatum. In the hippocampus, NT exerted a biphasic effect, behaving as a stimulator in the presence of 1 × 10 − 12 M–1 × 10 − 10 M concentration but as an inhibitor at 1 × 10 − 8 M–1 × 10 − 5 M concentration. In order to test the involvement of high-affinity NT receptor (NTS1) in NT inhibitory effect, assays were carried out in the presence of 1 × 10 − 6 M NT and/or SR 48692 (Sanofi-Aventis, U.S., Inc.), a specific antagonist for this receptor, dissolved in dimethylsulfoxide (DMSO) 10% v/v. As controls, membranes incubated with DMSO and/or NT 1 × 10 − 6 M plus DMSO were processed. It was found that NT + DMSO decreased [ 3H]-QNB binding to cerebral cortex, cerebellum and hippocampal membranes by 49%, 32% and 53%, respectively. This inhibition was not observed with the DMSO control group. Membrane preincubation with 1 × 10 − 6 M SR 48692 failed to alter NT effect on binding. SR 48692 at 1 × 10 − 6 M concentration decreased the binding by 50% only in cerebral cortex membranes, suggesting a possible direct effect of the antagonist on muscarinic receptors in this area. It was therefore concluded that the high-affinity NT receptor may not be involved in ligand binding inhibition to muscarinic receptor by NT.

Antidiabetic Effect of Essential Oil from Artemisia sieberi Growing in Jordan in Normal and Alloxan Induced Diabetic Rats

The aim of this study was to evaluate the effects of essential oil extracted from aerial parts of Artemisia sieberi in normal and alloxan induced diabetic rats. Fifty rats were divided into five groups of 10 each. Group I normal rats received 1 mL day(-1) of dimethyl sulfoxide (control); group II normal rats received a single dose (80 mg kg(-1) b.wt.) of essential oil extract of Artemisia sieberi; group III diabetic rats received 1 mL day-of dimethyl sulfoxide; group IV diabetic rats received the oil extract (80 mg kg(-1) b.wt.); group V diabetic rats received metformin (14.2 mg kg(-1) b.wt.). All treatments were orally administered once a day for six weeks. Changes in blood glucose concentration, body weight and food and water intake were measured and the data obtained were compared with that of metformin. The essential oil extract significantly (p < 0.05) lowered blood glucose level as well as food and water intake in diabetic rats accompanied by an increase in body weight gain with no apparent side effect when compared with untreated diabetic rats. These effects were found to be closely similar to that of metformin, a common antidiabetic drug. On other hand, no apparent improvement on body weight gain in diabetic rats treated with metformin. In addition, for all parameters measured, the oil extract showed no effect in normal rats. In conclusion, the essential oil of Artemisia sieberi exhibited antidiabetic activity in alloxan-induced diabetic rats. Present findings support the possible use of the essential oil of Artemisia sieberi as a remedy for diabetes mellitus in humans.

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.