Toll-like receptor 9 SNPs are susceptible to the development and progression of membranous glomerulonephritis: 27 years follow up in Taiwan

[[abstract]]Abstract The purpose of this study was to determine whether toll-like receptors 9 (TLR9) gene polymorphisms (rs352139 and rs352140) were markers of susceptibility to the development and progression of membranous nephropathy (MGN) in Taiwanese patients. The polymorphisms were investigated by polymerase chain reaction in 397 Taiwanese individuals (134 MGN patients and 263 controls). Patients with malignancy, chronic infectious diseases, lupus nephritis, or drug-induced secondary MGN were excluded from the study. Data showed AA genotype at rs352139 SNP or GG genotype at rs352140 SNP may indicate higher risk for MGN (odds ratio [OR] = 1.55; 95% confidence interval [CI] = 1.02-2.35, at rs352139 SNP; OR = 1.57; 95% CI = 1.03-2.39, at rs352140 SNP). However, MGN patients with A-G haplotype were susceptible for decreased creatinine clearance rate and for seriously tubule-interstitial fibrosis. The result suggests for the first time that TLR9 (rs352139 and rs352140) polymorphisms may contribute to the development and progression of MGN

Vasorelaxation Effects of 2-Chloroethanol and Chloroacetaldehyde in the Isolated Rat Aortic Rings

2-Chloroethanol (2-CE) is a commonly used solvent in industry; unfortunately, severe hypotension is one of main toxic signs during intoxication. Calcium ion modulation is considered to be an important role of vasorelaxation. The aim of this study is to evaluate either 2-CE or its main metabolite, chloroacetaldehyde (CAA), possible cause of hypotension, by using isolated rat aortic rings. Results revealed that 2- CE caused a weakly relaxation in the phenylephrine (PE) pre-induced endothelium-intact aortic rings. However, its metabolite, CAA induced vasorelaxation and showed dose dependency in endothelium-intact and - denuded aortic rings. The half inhibitory concentration (IC 50) of 2-CE exceeded 50 mM; meanwhile, the IC50 values of CAA in the endothelium- intact and -denuded aortic rings were 3.3 and 2.7 mM, respectively. The CAA-induced relaxation could be significantly attenuated by adding calcium (CaCl2) and various Ca2+ channel blockers, dantrolene, nifedipine, and NiCl2. Nifedipine presents the most strong inhibition effect among the calcium blockers. In conclusion. it is suggested that the hypotension effect of 2-CE intoxicated cases may be mainly mediated by its metabolite CAA, and calcium channels are partially involved inducing the vasorelaxation

Effects of chloroacetaldehyde on 2-chloroethanol intoxication and its related mechanisms

2-氯乙醇(2-chloroethanol)為一種廣泛使用的工業用溶劑。台灣部份農民會使用2-氯乙醇當作葡萄催芽劑讓葡萄樹芽快速發芽，因此臨床上偶有中毒的個案。目前未有有效的解毒方法，因此，2-氯乙醇的中毒機制及解毒方法確實仍有待進一步研究。本研究分為四個實驗: 試驗一：酒精去氫酶(alcohol dehydrogenase)與glutathione被認為與2-氯乙醇的代謝有關，因此擬以酒精去氫酶抑制劑fomepizole與影響Glutathione前驅物N-acetylcysteine來探討2-氯乙醇的毒理機制。結果顯示5 mg/kg fomepizole可以明顯降低2-氯乙醇的致死毒性，其半致死劑量由65.1 mg/kg提升至180 mg/kg。然而，醛類去氫酶(aldehyde dehydrogenase)抑制劑disulfiram卻會增強2-氯乙醇的致死毒性作用。另外，以fomepizole給予2-氯乙醇中毒的大鼠，明顯降低血漿中氯乙醛濃度，並且增加提升因2-氯乙醇而消耗的glutathione。合併給予N-acetylcysteine與fomepizole降低血漿中氯乙醛，並且回復因2-氯乙醇而消耗的Glutathione至正常值。以上結果顯示分解酵素在2-氯乙醇中毒可能扮演重要角色，因此，合併給予2-氯乙醇中毒的大鼠fomepizole與N-acetylcysteine具有保護作用。 試驗二：在臨床上發現2-氯乙醇中毒的病患具有嚴重低血壓的現象，鈣離子被認為是血管鬆弛主要因子。本研究以大鼠離體血管模式探討2-氯乙醇與氯乙醛對降低血壓的影響。結果顯示2-氯乙醇本身對離體大鼠血管並不會造成鬆弛的現象，其半抑制濃度大於50 mM；但是氯乙醛會造成明顯血管鬆弛，在無論是否有血管內皮細胞的存在均相似，其半抑制濃度分別為3.3 mM與2.7 mM。另外，給予鈣離子和鈣離子通道阻斷劑如dantrolene、nifedipine和NiCl2皆會抑制氯乙醛對血管鬆弛的作用，特別是nifedipine。以上結果顯示2-氯乙醇中毒所引起的低血壓是經由其代謝物氯乙醛所引起的，且與鈣離子通道有關。 試驗三：2-氯乙醇中毒常伴有心臟血管毒性，但是其機制仍未明。本實驗以大鼠離體心臟模式探討2-氯乙醇對心臟的毒性試驗。結果顯示2-氯乙醇會抑制左心房的跳動，但氯乙醛不但會抑制左心房的跳動且會造成孿縮作用。鈣離子通道阻斷劑nifidipine會降低左心房因氯乙醛所造成的跳動抑制與孿縮現象。另外，2-氯乙醇不會造成右心房的自行跳動停止，然而氯乙醛會造成右心房的跳動停止。在2-氯乙醇中毒大鼠的腦與分離的血管可以發現neuronal nitric oxide synthase蛋白有明顯的表現。心臟組織亦中可發現neuronal nitric oxide synthase和calmodulin蛋白在氯乙醛處理組的表現較2-氯乙醇處理組高，以nifedipine前處理可以降低因氯乙醛所誘發的neuronal nitric oxide synthase與calmodulin蛋白的表現。以上結果顯示2-氯乙醇中毒所造成的心臟毒性可能是經由氯乙醛所引起的，nifedipine可經由降低neuronal nitric oxide synthase蛋白的表現而達到保護的效果。 試驗四：有鑒於許多使用2-氯乙醇的葡萄農可能暴露在慢性中毒的危險，所以本實驗擬以安氏試驗法(Ames test)，染色體變異試驗(chromosome aberration test)及小鼠微核試驗(micronucleus assay)偵測2-氯乙醇及氯乙醛是否具有突變性。從安氏試驗法結果發現氯乙醛並未誘發TA98及TA100菌株。由染色體變異試驗結果發現2-氯乙醇並不會造成染色體變異，但是高劑量且經由小鼠肝臟酵素活化的氯乙醛會導致染色體變異頻率增加。經由小鼠微核試驗的結果發現2-氯乙醇並不會造成小鼠週邊血微核的增加，但是氯乙醛會造成小鼠週邊血微核的增加。結果顯示氯乙醛可能會造成致突變性，詳細的致突變性機制還需要更多的實驗來驗證。2-Chloroethanol (2-CE) is widely used industrial solvent. In Taiwan, farmers apply 2-CE on grapevines to accelerate grape growth, a practice that some cases have caused poisoning in humans. Thus there is strong interest in identifying antidotes to 2-CE. The aim of this study was contained with four parts. Part I, this study examines the protective role in 2-CE intoxicated rats. Alcohol dehydrogenase and glutathione were hypothesized to be important in the metabolism of 2-chloroethanol. This study used fomepizole, an alcohol dehydrogenase inhibitor, and chemicals that affected glutathione metabolism to study 2-CE toxicity. Notably, fomepizole 5 mg/kg significantly increased median lethal dose (LD50) of 2-CE from 65.1 mg/kg to 180 mg/kg, and reduced the production of a potential toxic metabolite chloroacetaldehyde (CAA) in animal plasma. In contrast, disulfiram (DSF), an aldehyde dehydrogenase inhibitor, increased the toxicity of 2-CE on the lethality in rats. Additional or pretreatment with N-acetylcysteine (NAC) and fomepizole significantly reduced plasma CAA concentrations. Fomepizole also significantly reduced 2-CE inhibited glutathione activity. Otherwise, pretreatment with NAC for four days followed by co-treatment with fomepizole significantly decreased formation of the metabolic CAA. These results indicated that its catalytic enzyme might play a vital role during 2-CE intoxication, and the combination of fomepizole and NAC could be a protective role in cases of acute 2-CE intoxication. Part II, severe hypotension is one of main toxic signs during intoxication. Calcium ion modulation is considered to be an important role of vasorelaxation. The aim of this study is to evaluate either 2-CE or its main metabolite, CAA, possible cause of hypotension, by using isolated rat aortic rings. Results revealed that 2-CE caused a weakly relaxation in the phenylephrine (PE) pre-induced endothelium-intact aortic rings. However, its metabolite, CAA induced vasorelaxation and showed dose dependency in endothelium-intact and -denuded aortic rings. The half inhibitory concentration (IC50) of 2-CE exceeded 50 mM; meanwhile, the IC50 values of CAA in the endothelium-intact and -denuded aortic rings were 3.3 and 2.7 mM, respectively. The CAA-induced relaxation could be significantly attenuated by adding calcium (CaCl2) and various Ca2+ channel blockers, dantrolene, nifedipine, and NiCl2. Nifedipine presents the most strong inhibition effect among the calcium blockers. In conclusion, it is suggested that the hypotension effect of 2-CE intoxicated cases may be mainly mediated by its metabolite CAA, and calcium channels are partially involved inducing the vasorelaxation. Part III, cardiovascular effects have often been found in 2-CE-intoxicated humans, but the mechanism by which 2-CE elicits cardiovascular toxicity is not clear. In this study an in vitro isolated rat atrium model was used to examine the cardiovascular toxicity of 2-CE. Results indicated that 2-CE inhibited tension in the isolated rat left atria. In addition, CAA caused significant tension inhibition and contracture in the isolated rat left atria. Nifedipine, an L-type calcium channel blocker, decreased CAA-induced tension inhibition and contracture. 2-Chloroethanol did not cause tension arrest in isolated rat right atria, but CAA did. Meanwhile, neuronal nitric oxide synthase (nNOS) was significantly expressed in 2-CE-intoxicated rat brain and isolated aortic rings. Furthermore, atrial nNOS and calmodulin (CaM) had significantly greater expression in the CAA group than the 2-CE group. Pretreatment with nifedipine decreased CAA-induced nNOS and CaM expression. These results indicate that 2-CE cardiovascular toxicity might be due to its metabolite CAA and that nifedipine is protective against nNOS-triggered cardiovascular toxicity. Part IV, 2-CE, and its metabolite, CAA was a well known mutagenicity agent. The genotoixic study of CAA had not been studied. Chronic occupation injury might escape the focus of 2-CE intoxication. In this study, we were using the in vitro and in vivo genotoxicity tests to examine the mutagenicity of 2-CE and CAA. First, the Ames test showed the 2-CE and CAA did not induce TA98 and TA100 Salmonella typhimurium strains reversions nearby the bacterial toxicity. Second, 2-CE did not induce chromosome aberrations formation by using Chinese ovary hamster cells, but CAA did induce chromosome aberrations formation at chromosome type gap aberration only after S9 activation. Third, 2-CE did not induce mice peripheral blood micronucleus formation, but high dosage of 2-CE (i.p. 1/2 LD50) would induce micronucleus formation. Otherwise, CAA induced micronucleus formation at low and high dosage (1/8 to 1/2 LD50). These results indicated that CAA is a genotoxic agent; however the mechanisms of CAA mutagenesis need further study.Contents Abstract in Chinese I Abstract IV Contents VIII Figure I. Study flow chart 1 PART I Protective effects of fomepizole on 2-chloroethanol toxicity 2 Abstract 3 Introduction 4 Materials and Methods 6 Chemicals 6 Animals 6 Determining the median lethality dose of 2-CE and the efficacy of potential antidotes in rats 6 The influence of CAA and glutathione on 2-CE toxicity 7 Measurement of plasma concentrations of CAA 7 Measurement of GSH activity in liver and kidney 8 Statistical analysis 9 Results 10 Effects of fomepizole, DSF on 2-CE induced lethality in rats 10 Effects of fomepizole, DSF on plasma CAA concentrations 10 Effects of fomepizole, DSF on 2-CE depletion GSH in liver 11 Discussion 12 Conclusion 14 References 15 Table 1-1 19 Table 1-2 20 Figure 1-1 21 Supplementary Table1-1 22 PART II Vasorelaxation effects of 2-chloroethanol and chloroacetaldehyde in the isolated rat aortic rings 23 Abstract 24 Introduction 25 Materials and Methods 27 Chemicals 27 Animals 27 Preparation and treatment of aortic rings 27 Experiment for effects of 2-CE and CAA in aortic rings of rats 28 Experiment for effects of CAA -induced relaxation with/without calcium 29 Experiment for Ca2+ channel blockers in CAA relaxed aortic ring of rats 29 Statistical analysis 30 Results and Discussion 31 2-CE and CAA relaxed aortic rings 31 CaCl2 attenuation on CAA relaxed aortic rings 32 Ca2+ channel blockers reduce CAA-induced vasorelaxation 33 References 36 Figure 2-1 40 Figure 2-2 41 Table 2-1 42 Figure 2-3 43 Figure 2-4 44 Figure 2-5 45 Figure 2-6 46 PART III Protective effects of nifedipine in chloroacetaldehyde-induced cardiovascular toxicity 47 Abstract 48 Introduction 49 Materials and methods 51 Chemicals 51 Animals 51 Western blotting for brain nNOS 51 Preparation and treatment of the isolated rat atria 52 Immunohistochemistry for nNOS in aortic rings and atria 53 Statistical analysis 54 Results 55 nNOS expression in 2-CE-intoxicated rat brain and aortic rings 55 Effects of 2-CE and CAA in isolated rat left atria 55 Effects of nifedipine on CAA-induced tension inhibition and contracture in isolated rat left atria 56 Effects of 2-CE and CAA in isolated rat right atria 56 nNOS and CaM expression in 2-CE, CAA and nifedipine pre-treated atria 56 Discussion 58 References 60 Figure 3-1 65 Figure 3-2 66 Table 3-1 67 Figure 3-3 68 Figure 3-4 69 Figure 3-5 70 Figure 3-6 71 PART IV Study of genotoxicity of 2-chloroethanol and chloroacetaldehyde using in vitro and in vivo tests 72 Abstract 73 Introduction 74 Materials and Methods 75 Ames test 75 Chromosome aberration test 76 Micronucleus assay 77 Statistical analysis 77 Results 78 Ames test 78 Chromosome aberration test 78 Micronucleus assay 78 Discussion 80 Reference 82 Figure 4-1 86 Figure 4-2 87 Table 4-1 88 Figure 4-3 89 Table 4-2 90 Figure 4-4 91 Table 4-3 92 Summary 9

Trends of coffee clubbing in Singapore.

This project is undertaken to investigate the market potential of coffee clubs in Singapore. In addition, an in-depth analysis of the major players operating in the local coffee club scene will also be carried out. We strongly believe that the results will provide valuable insights into the present coffee club scene in Singapore. The resulting effort will be a marketing plan that has been devised as part of the proposal to establish a brand new coffee club to top this growing market

Comparison of Genotoxicity and Pulmonary Toxicity Study of Modified SiO₂ Nanomaterials

Surface-modified nano-SiO2 is a common additive in many products. However, the safety of nano-SiO2 products under various modifications is still unclear. In this study, we investigated the genotoxicity and acute pulmonary toxicity of nano-SiO2 with or without modification. The samples used in this study included: sample A (SA, 55.16 nm, 411.3 mg/mL), modified sample A (mSA, 82.29 nm, 37.7 mg/mL), sample B (SB, 22 nm, 358.0 mg/mL), and modified sample B (mSB, 86.64 nm, 37.7 mg/mL). In the genotoxicity study, we conducted an Ames test, chromosomal aberration test (CA), and a micronucleus (MN) test. The SA, mSA, and mSB groups showed negative results in all these genotoxicity tests. Only SB showed a weakly positive reaction in these assays, but the genotoxicity could be reversed after S9 metabolism or modification. In the acute pulmonary toxicity test, the rats were given an intratracheal instillation (IT) (0.5 mL/kg) of diluted samples and sacrificed after 1 or 14 days. The mortality rate, number of leukocytes and cytokines of TNF-α in the bronchoalveolar lavage fluid (BALF), and the pathology in the lungs were determined. The results revealed that mSA posed acute toxicity in rats. After modification, the pulmonary toxicity was increased in mSA but decreased in mSB on Day 1, and no significant difference was observed on Day 14. In conclusion, there was no observed genotoxicity in either SA or SB, while mSA posed acute inhalation toxicity to rats that decreased in mSB after modification. This indicates that the decrease in pH level in SA and decrease in the solid content in SB are considered after the trifluorosilane surface-modified amorphous nano-silica

Comparison of Genotoxicity and Pulmonary Toxicity Study of Modified SiO2 Nanomaterials

Surface-modified nano-SiO2 is a common additive in many products. However, the safety of nano-SiO2 products under various modifications is still unclear. In this study, we investigated the genotoxicity and acute pulmonary toxicity of nano-SiO2 with or without modification. The samples used in this study included: sample A (SA, 55.16 nm, 411.3 mg/mL), modified sample A (mSA, 82.29 nm, 37.7 mg/mL), sample B (SB, 22 nm, 358.0 mg/mL), and modified sample B (mSB, 86.64 nm, 37.7 mg/mL). In the genotoxicity study, we conducted an Ames test, chromosomal aberration test (CA), and a micronucleus (MN) test. The SA, mSA, and mSB groups showed negative results in all these genotoxicity tests. Only SB showed a weakly positive reaction in these assays, but the genotoxicity could be reversed after S9 metabolism or modification. In the acute pulmonary toxicity test, the rats were given an intratracheal instillation (IT) (0.5 mL/kg) of diluted samples and sacrificed after 1 or 14 days. The mortality rate, number of leukocytes and cytokines of TNF-α in the bronchoalveolar lavage fluid (BALF), and the pathology in the lungs were determined. The results revealed that mSA posed acute toxicity in rats. After modification, the pulmonary toxicity was increased in mSA but decreased in mSB on Day 1, and no significant difference was observed on Day 14. In conclusion, there was no observed genotoxicity in either SA or SB, while mSA posed acute inhalation toxicity to rats that decreased in mSB after modification. This indicates that the decrease in pH level in SA and decrease in the solid content in SB are considered after the trifluorosilane surface-modified amorphous nano-silica

AQP4 Attenuated TRAF6/NFκB Activation in Acrylamide-Induced Neurotoxicity

Acrylamide (ACR) is present in high-temperature-processed high-carbohydrate foods, cigarette smoke, and industrial pollution. Chronic exposure to ACR may induce neurotoxicity from reactive oxygen species (ROS); however, the mechanisms underlying ACR-induced neurotoxicity remain unclear. We studied 28-day subacute ACR toxicity by repeatedly feeding ACR (0, 15, or 30 mg/kg) to rats. We conducted RNA sequencing and Western blot analyses to identify differences in mRNA expression in the blood and in protein expression in the brain tissues, respectively, of the rats. AQP4 transient transfection was performed to identify potential associations with protein regulation. The rats treated with 30 mg/kg ACR exhibited hind-limb muscle weakness. Matrix metalloproteinase (MMP9) expression was higher in the ACR-treated group than in the control group. ACR induced MMP-9 and AQP4 protein expression in the brain tissues of the rats, which subsequently presented with neurotoxicity. In the in vitro study, Neuro-2a cells were transiently transfected with AQP4, which inhibited MMP-9 and TNF receptor-associated factor 6 (TRAF6) expression, and inhibited ACR induced expression of TRAF6, IκBα, and nuclear factor κB (NFκB). Using a combination of in vivo and in vitro experiments, this study revealed that depressive symptoms associated with ACR-induced neurotoxicity are associated with downregulation of AQP4 and induction of the TRAF6 pathway

NT5C2 methylation regulatory interplay between DNMT1 and insulin receptor in type 2 diabetes

Epigenetics alternation of non-genetic variation and genome-wide association study proven allelic variants may associate with insulin secretion in type 2 diabetes (T2D) development. We analyzed promoter DNA methylation array to evaluate the associated with increased susceptibility to T2D (30 cases, 10 controls) and found 1,091 gene hypermethylated in promoter regions. We performed the association study of T2D and found 698 single nucleotide polymorphisms in exon and promoter sites by using 2,270 subjects (560 cases, 1,710 controls). A comparison of DNA hypermethylation and gene silencing of mouse T2D results in our T2D patients’ results showed that the 5′-nucleotidase, cytosolic II (NT5C2) and fucosyltransferase 8 (FUT8) genes were strongly associated with increased susceptibility to T2D. DNA hypermethylation in promoter regions reduced NT5C2 gene expression, but not FUT8 in T2D patients. NT5C2 protein expression was decreased in pancreatic β-cells from T2D mice. Transient transfection NT5C2 into RIN-m5F cells down-regulated DNA methyltransferase I (DNMT1) expression and up-regulation of the insulin receptor. Moreover, NT5C2 knockdown induced in DNMT1 overexpression and insulin receptor inhibition. Taken together, these results showed that NT5C2 epigenetically regulated insulin receptor in patients and mice with T2D, and maybe provide for T2D therapy strategy

Anti-Oxidant, Anti-Mutagenic Activity and Safety Evaluation of Antrocin

Antrocin is a novel compound isolated from Antrodia cinnamomea, and is classified as a sesquiterpene lactone. The therapeutic efficacy of antrocin has been studied, and it has shown an antiproliferative effect on various cancers. The aim of this study was to evaluate the anti-oxidant activity, potential genotoxicity, and oral toxicity of antrocin. Ames tests with five different strains of Salmonella typhimurium, chromosomal aberration tests in CHO-K1 cells, and micronucleus tests in ICR mice were conducted. The results of anti-oxidant capacity assays showed that antrocin has great anti-oxidant activity and is a moderately strong antimutagenic agent. In the results of the genotoxicity assays, antrocin did not show any mutagenic potential. In the 28-day oral toxicity test, Sprague Dawley rats were gavaged with 7.5 or 37.5 mg/kg of antrocin for 28 consecutive days. In addition, 7.5 mg/kg sorafenib, an anti-cancer drug, was used as a positive control for toxicity comparison. At the end of the study, antrocin did not produce any toxic effects according to hematology, serum chemistry, urine analysis, or histopathological examinations. According to the results of the genotoxicity and 28-day oral toxicity study, antrocin, at a dose of 37.5 mg/kg, did not cause adverse effects and can be a reference dose for therapeutic agents in humans