Effects of 2,3,7,8-TCDD and PCB 126 on human thymic epithelial cells in vitro

2,3,7,8-Tetrachloro-dibenzo- p-dioxin (TCDD) is a ubiquitously distributed xenobiotic. The adverse effects of TCDD on the mammalian immune system have been studied for decades, but it is still unclear whether TCDD has direct effects on T-lymphocytes or whether it acts via the thymic microenvironment. We have studied the effects of TCDD on primary cultures of human thymic epithelial cells (TEC) focusing on differentiation markers, integrins and adhesion molecules involved in cell-cell and in cell-matrix interactions. TEC were treated with TCDD at concentrations of 0.001, 0.01, 0.1, 1.0 or 10.0 nM or with 100 nM PCB 126 (3,3',4,4',5-pentachlorobiphenyl) for 3 days, and were then analysed by flow cytometry for expression of surface antigens using monoclonal antibodies against Hassall's bodies (TE-8, TE-16) or against surface structures such as CD29, CD49b, CD49e, CD49f, CD51, CD54, CD58, CD61 and CD106. At TCDD concentrations as low as 0.01 nM we found a significant increase in terminally differentiated, TE-16-positive TEC; at a ten-fold greater concentration the number of cells marked with the TE-8 antibody was also increased. With both markers the most pronounced effect (approximately +15%) was observed at 1 nM TCDD. An increase of cells expressing the integrin alpha-chains CD49b, CD49e and CD51 as well as CD54 was observed at concentrations of 0.1 nM TCDD or higher. The proportion of cells expressing CD106 or CD49f decreased significantly upon treatment with TCDD. No effects on the integrin beta-chains CD29 and CD61 could be detected. Overall, PCB 126 induced similar changes to TCDD. In summary, TCDD and a coplanar PCB induced terminal differentiation of human TEC along with changes of integrins and other adhesion molecules. These receptors and their interplay with the extracellular matrix have key functions in the maturation of T-lymphocytes and it is plausible that their alteration would be involved in TCDD-induced immunotoxicity.     

Effects of OCT2 c.602C > T genetic variant on the pharmacokinetics of lamivudine

Abstract

1. The renal excretion of organic cation drugs, including lamivudine, is mostly mediated by OCT2 in vitro. To date, three putatively relevant single nucleotide polymorphisms (SNPs), including c.596C?>?T (p.Thr199Ile), c.602C?>?T (p.Thr201Met), and c.808G?>?T (p.Ala270Ser) have been observed in Asians. The effects of the SLC22A2 c.602C?>?T genetic variant on the pharmacokinetics of lamivudine were studied with healthy Korean subjects.

2. Nineteen healthy subjects carrying either the SLC22A2 c.602CC (n?=?12) or c.602CT (n?=?7) genotype volunteered for this study. A single 100?mg dose of lamivudine was orally administered to each subject. Blood samples were collected for up to 24?h and the plasma concentrations of lamivudine were measured using liquid chromatography-tandem mass spectrometry.

3. The mean plasma concentration–time profiles of lamivudine in the c.602CC and c.602CT genotype groups were similar. There was no significant difference in the overall pharmacokinetic parameters of lamivudine between the c.602CC and c.602CT genotype groups. Differences in renal clearance and tubular secretion clearance were also not statistically significant between the two genotype groups.

4. The SLC22A2 c.602C?>?T genotype did not affect the pharmacokinetics of lamivudine in humans in vivo. Dose adjustment of lamivudine is not required between individuals with c.602CC and c.602CT genotypes.     

全反式维甲酸诱导小鼠间充质干细胞C3H10T1/2表达碱性磷酸酶

目的分析全反式维甲酸对小鼠间充质干细胞C3H10T1/2表达碱性磷酸酶的影响。方法全反式维甲酸（10-6M）、维甲酸核受体RAR的激动剂Ch55与MAPK信号通路的阻滞剂处理C3H10T1/2细胞,钙钴染色检测碱性磷酸酶活性,定量RT-PCR方法检测量效关系以及时相分布;采用萤光素酶报告基因检测碱性磷酸酶的启动子是否接受维甲酸的调控。结果维甲酸处理C3H10T1/2细胞24h即能够明显上调ALP的活性,具有明显的剂量依赖特性,与RAR有关,无MAPK信号通路的参与,在其转录起始位点上游-512bp的范围内检测到核受体RAR的调控位点。结论维甲酸能够通过核受体RAR直接上调碱性磷酸酶的转录水平。     

Effects of 1α,25-dihydroxyvitamin D3 on transport and metabolism of adefovir dipivoxil and its metabolites in Caco-2 cells

Effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), natural ligand of the VDR, on the fates of adefovir dipivoxil (P-gp substrate) and its metabolites, mono(POM)-PMEA and adefovir (MRP4 substrate), were investigated in Caco-2 cells. After 1,25(OH)2D3-treatment, higher apical efflux of adefovir was observed after a 60 min incubation of adefovir divipoxil. Changes in these washout studies were predicted by a catenary model for the Caco-2 monolayer that described a higher MRP4 activity with 1,25(OH)2D3 treatment, as confirmed by Western blotting. Moreover, 1,25(OH)2D3 treatment (100 nM for 3 days) resulted in increased basolateral (B) to apical (A) (B-to-A) transport of adefovir dipivoxil but an unchanged A-to-B flux, rendering an elevated efflux ratio (EfR) (from 1.97 to 3.19). The EfR values in control and 1,25(OH)2D3-treated groups in these transport studies were reduced to 1.32 and 1.57, respectively, in the presence of verapamil (50 μM), the P-gp inhibitor. The B-to-A transport of the metabolite, adefovir, was increased in 1,25(OH)2D3-treated cells in the presence of verapamil, whereas the A-to-B and B-to-A transport of mono(POM)-PMEA remained unchanged. But the verapamil and 1,25(OH)2D3 treatments failed to alter rates of sequential metabolism of adefovir dipivoxil in cell lysate. The composite data established that 1,25(OH)2D3 treatment increased both P-gp and MRP4 transport activities without affecting the metabolism of adefovir dipivoxil by esterases. Moreover, an asymmetric appearance of metabolites, being higher with apical application, was observed. According to the catenary model, the asymmetry is suggestive that esterases are predominantly localized on the apical membrane and within the cell.     

Effects of squalene on expression of LDLR in HepG2 cells and its mechanism北大核心CSCD

Aim To investigate the effect of squalene on LDLR expression in HepG2 cells and its mechanism of down-regulated cholesterol. Methods The proliferation of HepG2 cells exposed to squalene at different concentrations was measured by MTT assay. The effect of squalene on the expression of LDLR in HepG2 cells was measured by flow cytometry and fluorescence mi-croscopy. The effect of different concentrations of squalene on the interaction between SCAP and Insig2, two key protein molecules of SREBP pathway, was assayed by FRET technology. Results MTT results showed that squalene had inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner. Flow cytometry and fluorescence microscopy results showed that squalene enhanced LDLR expression in HepG2 cells compared with the control group. The results of FRET technology revealed that compared with model control group, the YFP fluorescence value in Squalene group dramatically declined, and the YFP fluorescence value of each drug group decreased with the range of 5-25 |xmol L1 squalene concentration. Conclusions Squalene may promote the expression of LDLR in HepG2 cells through inhibiting the interaction between SCAP and Insig2 proteins in SREBP pathway, which may confirm that squalene is a potential novel drug for the down-regulation of cholesterol level. © 2018 Publication Centre of Anhui Medical University. All rights reserved.     

Effects of Ezetimibe/Simvastatin 10/20 mg vs. Atorvastatin 20 mg on Apolipoprotein B/Apolipoprotein A1 in Korean Patients with Type 2 Diabetes Mellitus: Results of a Randomized Controlled Trial

Background

Although the efficacy of ezetimibe/simvastatin and atorvastatin on traditional lipid parameters has been studied extensively, the apolipoprotein B/apolipoprotein A1 (ApoB/ApoA1) ratio, which has a better predictive value for cardiovascular events, has not previously been used as a primary endpoint in these two treatment groups.

Objective

Our objective was to compare the efficacy and safety of ezetimibe/simvastatin 10/20 mg versus atorvastatin 20 mg once daily in Korean patients with type 2 diabetes mellitus.

Study Design

This study was an open-label, randomized, controlled study. Type 2 diabetes patients with high levels of low-density lipoprotein (LDL) cholesterol (>100 mg/dL) were randomized to receive ezetimibe/simvastatin or atorvastatin.

Main Outcome Measure

The primary endpoint was the difference in the percent change of ApoB/ApoA1 at 12 weeks, and secondary endpoints were changes in lipid profiles, glycosylated hemoglobin (HbA_1c), homeostatic model assessment (HOMA) index, and C-reactive protein.

Results

In total, 132 patients (66 for each group) were enrolled and randomized. After 12 weeks of treatment, the ApoB/ApoA1 ratio was significantly reduced in both groups; however, the difference of changes between the two groups was not statistically significant (ezetimibe/simvastatin ?38.6 ± 18.0 % vs. atorvastatin ?34.4 ± 15.5 %; p = 0.059). There were no significant differences in changes to total cholesterol, LDL cholesterol, high-density lipoprotein cholesterol, triglycerides, ApoB, and ApoB48 between the two groups. However, the increments of ApoA1 were significantly greater in the ezetimibe/simvastatin group than in the atorvastatin group (2.8 ± 10.0 vs. ?1.8 ± 9.8 %; p = 0.002). In the per-protocol analysis, improvement in ApoB/ApoA1 was significantly greater in the ezetimibe/simvastatin group (?42.8 ± 11.8 vs. ?36.7 ± 13.2 %; p = 0.019). The changes in HbA_1c, HOMA index, and C-reactive protein were comparable between the two groups. The adverse reaction rate was similar between the two groups (24.2 vs. 34.9 %; p = 0.180).

Conclusion

Ezetimibe/simvastatin 10/20 mg is comparable to atorvastatin 20 mg for the management of dyslipidemia, and may have more favorable effects on apolipoprotein profiles than atorvastatin 20 mg in Korean patients with type 2 diabetes mellitus.     

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10⁻⁷ molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.     

PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells

Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100 μM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1 μM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50 μM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1 μM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.     

The inhibition of HIF-2α on the ATM/Chk-2 pathway is involved in the promotion effect of arsenite on benzo(a)pyrene-induced cell transformation

Both arsenite and benzo(a)pyrene (BaP) are known human carcinogens. Studies on the mode-of-action of arsenite indicate that it can also act as co-carcinogen or as a cancer promoter, and that it can facilitate progression of cancers. Some studies on development of lung cancers have suggested a synergism between arsenite exposure and cigarette smoking. The mechanism of action for such an effect, however, remains obscure. In the present study, we investigated the effects of HIF-2α on arsenite- and BaP-induced cell malignant transformation as well as on signal transduction pathways in human bronchial epithelial (HBE) cells. The results show that arsenite accelerates the neoplastic transformation and migration of cells and enhances chromosomal aberrations induced by BaP. HIF-2α is involved in blocking the effects of arsenite in activating the ATM/Chk-2 pathway and in repair of DNA damage induced by BaP. Moreover, blocking of HIF-2α prevents the effects of arsenite on the neoplastic transformation, cell migration, and chromosomal aberrations caused by BaP. These results indicate that the repressive effect of HIF-2α on the ATM/Chk-2 pathway leads to genomic instability, which is involved in arsenite-accelerated, BaP-induced malignant transformation of HBE cells.     

Effects of rutin on the expression of PPARγ in skeletal muscles of db/db mice

The effect of rutin (RT) on the expression of PPARγ in the skeletal muscle of db/db mice has been investigated. Thirty db/db mice were randomized into 5 equal groups: model control group (MCG), positive control group 30 mg/kg pioglitazone hydrochloride (PCG), low-dose rutin group 50 mg/kg (LRT), middle-dose rutin group 100 mg/kg (MRT), high-dose rutin group 200 mg/kg (HRT), and 6 other db/m mice were used as the normal control (NC). The expression of PPARγ mRNA and protein in the skeletal muscle was determined by real-time quantitative PCR and Western Blot, respectively. Treatment with HRT resulted in significant decreases in the plasma levels of glucose and lipids (p < 0.001). Compared to the MCG, the expression of PPARγ mRNA and protein of the skeletal muscle was significantly increased in the HRT and PCG groups (p < 0.001 and p < 0.05, respectively). Rutin can increase the expression of PPARγ in skeletal muscles of db/db mice.     

Effects of tetrandrine on cytosolic free calcium in cultured bovine smooth muscle cells

AIM: To study the effects of tetrandrine (Tet) on smooth muscle cells (SMC). METHODS: Using Fura 2-AM and AR-CM-MIC cation measurement system, cytosolic free calcium ([Ca~(2 )]_i) was examined in cultured bovine SMC.     

Genotoxicity of cadmium in marine diatom Chaetoceros tenuissimus using the alkaline Comet assay

Genotoxic effects of cadmium on phytoplankton Chaetoceros tenuissimus have been evaluated using DNA damage by Comet assay. Cadmium concentrations ranging from 2.4 to 10 mg/l were used to evaluate the effects. Results showed that as the concentration of Cd increased growth of the diatom decreased. Alkaline single-cell gel electrophoresis (Comet assay) method, which is highly sensitive in detection of DNA damage in eukaryotic cells, was used to observe genomic changes in marine diatom cells. DNA damage was measured as percent number of comets and normal cells. 65% cells were found to be damaged at 10 mg/l concentration of Cd as compared to 23% in 2.4 mg/l and only 5% in controls. More than 50% apoptic cells were observed on 8th day at 10 mg/l and 12th day at 7.5 mg/l concentrations. At lower Cd concentrations (4.5 mg/l and below) the damage was below 30% till the last day. This suggested that higher Cd levels have early damaging effects on cell nuclear material and that % injury increases with advancement of exposure period. One advantage of use of C. tenuissimus is the ease with which it can be cultured in a defined medium. C. tenuissimus diatom can be used as an in vivo model for ecogenotoxicity assessment using the Comet assay.Special Issue on Biomarkers of Marine Pollution and Bioremediation     

Effects of carnosine on NMDA -induced necrosis in NGF- differentiated PC12 cells

The purpose of this study was to assess the effects of carnosine on N - methyl - D - aspartate acid （NMDA）-induced necrosis in NGF - differentiated PC12 rat pheoehromocytoma cells. Within 30 min of 2mM NMDA insults, 40% cells died by necrosis. Carnosine reversed the neurotoxicity in a time-dependent and concentration-dependent manner. After 18 hours application of carnosine, it showed a peak protection at a concentration of 5 raM. This protection was antagonized by histamine H1 receptor antagonist pyrilamine,     

Effects of verapamil on the cardiac α1-adrenoceptor signalling system in diabetic rats

We evaluated the effects of chronic verapamil treatment on the cardiac α₁-adrenoceptor signalling system in streptozocin-induced diabetic rats. The decrease in maximum cell surface [³H]bunazosin binding (B_max) in isolated cardiac myocytes from the diabetic group (−46%, P < 0.01) was completely reversed by a 4-week course of verapamil, while B_max in the verapamil-treated control group was unchanged. Similarly, the reduction in ventricular inositol 1,4,5-trisphosphate (IP₃) production after stimulation with 10 μM noradrenaline (NA) seen in diabetes (−30%, P < 0.01) was completely normalized by verapamil, while the response in the verapamil-treated control group was unaffected. These results indicate that verapamil can induce complete recovery of the impaired cardiac α₁-adrenoceptor signalling system in the diabetic heart without affecting glucose metabolism.     

Involvement of HIF-2α-mediated inflammation in arsenite-induced transformation of human bronchial epithelial cells

Arsenic is a well established human carcinogen that causes diseases of the lung. Some studies have suggested a link between inflammation and lung cancer; however, it is unknown if arsenite-induced inflammation causally contributes to arsenite-caused malignant transformation of cells. In this study, we investigated the molecular mechanisms underlying inflammation during neoplastic transformation induced in human bronchial epithelial (HBE) cells by chronic exposure to arsenite. The results showed that, on acute or chronic exposure to arsenite, HBE cells over-expressed the pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β). The data also indicated that HIF-2α was involved in arsenite-induced inflammation. Moreover, IL-6 and IL-8 were essential for the malignant progression of arsenite-transformed HBE cells. Thus, these experiments show that HIF-2α mediates arsenite-induced inflammation and that such inflammation is involved in arsenite-induced malignant transformation of HBE cells. The results provide a link between the inflammatory response and the acquisition of a malignant transformed phenotype by cells chronically exposed to arsenite and thus establish a previously unknown mechanism for arsenite-induced carcinogenesis.     

Effects of stilbenes on glucose and lipid metabolism in insulin resistant HepG2 cells北大核心CSCD

Aim To explore the effect of four stilbenes including rhaponticin, desoxyrhaponticin, rhapontigenin and resveratrol on glucose and lipid metabolism in insulin-resistant HepG2 cells induced by high glucose and high fat. Methods The model of insulin resistance was established by incubating HepG2 cells with a complex of glucose and oleic acid. MTT assay was used to detect cell viability. The intracellular triglyceride (TG) and glucose levels were measured by the kit method. The lipid production was observed by oil red O staining, and the cell morphology and uptake of 2-NBDG were observed by confocal microscope. The PPAR signaling pathway and PI3K/Akt insulin signaling pathway related proteins were determined by Western blot to evaluate the effect of stilbenes on glycolipid metabolism in IR-HepG2 cells. Results The complex containing 50 mmol • L-1 glucose and 0.5 mmol • L-1 oleic acid induced a successful insulin resistance model in HepG2 cells for 48 hours. Compared with the model group, all the four compounds could reduce the content of TG and lipid accumulation, and increase the ability of glucose uptake. These compounds could significantly inhibit the expression level of peroxisome proliferatorsactivated receptor (PPAR-) lipid metabolism-related proteins, and up-regulate the expression of phosphoinositide 3 -kinase (PI3 K) glycometabolism-related proteins, among which rhaponticin and resveratrol had the most significant effect. Conclusions The four stilbenes may alleviate insulin resistance in HepG2 cells by regulation of glucose and lipid metabolism disorder, especially rhaponticin and resveratrol. The mechanism may be related to the regulation of PPAR signaling pathway and the activation of PI3 K/AKT/mTORl pathway to reduce lipid production and promote glucose uptake. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Bmal1 inhibits phenotypic transformation of hepatic stellate cells in liver fibrosis via IDH1/α-KG-mediated glycolysis

Hepatic stellate cells(HSCs)play an important role in the initiation and development of liver fibrogenesis,and abnormal glucose metabolism is increasingly being...