Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin‐LR in human laryngeal epithelial cells (Hep‐2)

The major toxic mechanism of Microcystin‐LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin‐LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep‐2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton‐associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep‐2. A dose‐dependent change in p‐Tau and p‐Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep‐2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 890–903, 2017.     

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate‐coated AgNPs reduced cell viability in a dose‐dependent manner. The IC50 values of silver ions and citrate‐coated AgNPs were 0.5 and 50 mg L^?1, respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679–692, 2016.     

Modulation of bile acids induced by paraquat in rabbits

Rabbit bile was examined for changes in composition induced by paraquat. Paraquat was administered intraperitoneally and changes in bile components were monitored by high performance liquid chromatography. Alterations in the ratios of total glycine/taurine conjugated bile acids (TGC/TTC), cholic acid/deoxycholic acid (CA/DC), cholic acid/chenodeoxycholic acid (CA/CDC) and cholic acid/cholesterol (CA/CH) were measured as an index of paraquat toxicity. A statistically significant increase in the ratio of TGC/TTC was observed, while CA/DC, CA/CDC and CA/CH showed a decrease. Phospholipids, protein, sugar, bilirubin, beta-carotene, vitamin A and vitamin E in the bile and serum of the experimental animals were also monitored. In bile, the levels of cholesterol, phospholipids, protein, sugar, and total bile acids increased while the levels of the antioxidants beta-carotene, vitamin A and vitamin E decreased. A decrease in the bilirubin content of the bile was also observed. These modifications may be useful clinically for assessment of paraquat toxicity.     

The maternal pheromone and bile acids in the lactating rat

Bile was drawn from virgin rats and from postpartum rats that were with young for 5, 12, 21, and 30 days, respectively. The bile thus drawn was analyzed enzymatically after chromatographic separation to test an hypothesis relating cholic acid and one of its metabolites, deoxycholic acid, to the appearance of the maternal pheromone. Our finding that cholic acid, but not deoxycholic acid, reached a peak that was tied specifically to the period of pheromonal emission led us to advance a revised hypothesis. We now think that cholic acid alone, or more likely a cholic metabolite other than deoxycholic acid, underlies the appearance of the pheromone.     

Albumin modulates docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma cell lines

Fish oil-containing diets rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) provide protection against tumorigenesis. The mechanisms of the cytotoxic effects of DHA include the production of reactive oxygen species (ROS). Albumin has antioxidant property and binds fatty acids, it may protect the cells against the DHA-induced cytotoxicity. In this study, we compared the susceptibility of three human hepatocellular carcinoma (HCC) cell lines (HepG2, Hep3B, Huh7) to the cytotoxic effects of DHA, and examined the changes in the susceptibility following albumin overexpression using transfection vectors or albumin downregulation using small interfering RNA (siRNA). HepG2 cells were the most susceptible to DHA-induced cytotoxicity and increased oxidative activities by DHA compared to Hep3B and Huh7 cells. The cytotoxic effects of DHA were concentration-dependently abrogated by typical antioxidants, a radical scavenger, an iron chelator and incubation with exogenous albumin. Overexpression of albumin in HepG2 cells markedly attenuated DHA-induced oxidative activities and cytotoxicity. Furthermore, knockdown of albumin in both Hep3B and Huh7 cells significantly enhanced the effects of DHA. The results of our in vitro experiments indicate that the cytotoxic effects of DHA on HCC cell lines are modulated by albumin.     

Antibody-mediated inhibition of fibroblast growth factor 19 results in increased bile acids synthesis and ileal malabsorption of bile acids in cynomolgus monkeys

Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-β) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.     

The Influence of 2-Hydroxypropyl-β-cyclodextrin on the Haemolysis Induced by Bile Acids

Cyclodextrins improve the water-solubility of drugs and can mask their haemolytic effect in parenteral use. Because the mechanism by which bile acids induce haemolysis is poorly understood, it has been investigated in the presence of 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD). The haemolytic effect of 1.8 mm solutions of cholic acid, chenodeoxycholic acid (CDCA), deoxycholic acid and ursodeoxycholic acid (UDCA) in isotonic buffer at pH 7.4 was investigated at 37°C in the presence of HP-β-CyD at concentrations from 0.18 to 32 mm . No haemolytic effect was evident for cholic acid and UDCA. The haemolytic effect of the other bile acids was reduced by addition of HP-β-CyD and was prevented at a molar ratio of 1:1 owing to complex formation. An HP-β-CyD:bile acid molar ratio greater than 5:1 had a different effect on the erythrocyte membrane, irrespective of the identity of the bile acid; the effect was in accordance with the complexion affinities. In the absence of HP-β-CyD, the haemolytic effect of CDCA and deoxycholic acid appeared related to their capacity to form a surface monolayer and to solubilize the components of the erythrocyte membrane. The haemolytic effect observed after complexation of the bile acids appeared to be solely the effect of HP-β-CyD, which was able to form a reversible inclusion complex with lipophilic components of the erythrocyte membranes at concentrations higher than 12 mm .     

First total synthesis of protoapigenone and its analogues as potent cytotoxic agents

Protoapigenone (1), isolated from Thelypteris torresiana, previously showed significant cytotoxic activity against five human cancer cell lines. In a continued structure-activity relationship study, the first total synthesis and modification of 1 were achieved. All synthesized compounds and related intermediates were evaluated for cytotoxic activity against five human cancer cell lines, HepG2, Hep3B, MDA-MB-231, MCF-7, and A549. Among them, 24 showed 2.2-14.2-fold greater cytotoxicity than 1 and naphthyl A-ring analogues remarkably enhanced the activity.     

Co-exposure to silver nanoparticles and cadmium induce metabolic adaptation in HepG2 cells

Although multiple studies have reported the toxicological effects and underlying mechanisms of toxicity of silver nanoparticles (AgNP) in a variety of organisms, the interactions of AgNP with environmental contaminants such as cadmium are poorly understood. We used biochemical assays and mass spectrometry-based proteomics to assess the cellular and molecular effects induced by a co-exposure of HepG2 cells to AgNP and cadmium. Cell viability and energy homeostasis were slightly affected after a 4-h exposure to AgNP, cadmium, or a combination of the two; these endpoints were substantially altered after a 24-h co-exposure to AgNP and cadmium, while exposure to one of the two contaminants led only to minor changes. Proteomics analysis followed the same trend: while a 4-h exposure induced minor protein deregulation, a 24-h exposure to a combination of AgNP and cadmium deregulated 43% of the proteome. The toxicity induced by a combined exposure to AgNP and cadmium involved (1) inactivation of Nrf2, resulting in downregulation of antioxidant defense and proteasome-related proteins, (2) metabolic adaptation and ADP/ATP imbalance, and (3) increased protein synthesis possibly to reestablish homeostasis. The adaptation strategy was not sufficient to restore ADP/ATP homeostasis and to avoid cell death.     

Comparison of the choleretic properties of bile acids

The choleretic properties of cholic, chenodeoxycholic, and deoxycholic acid and their taurine and glycine conjugates were compared to their ability to form micelles. It has previously been concluded that deoxycholate has the lowest critical micellar concentration; chenodeoxycholate is slightly higher and cholic is much higher. Conjugation with glycine and taurine has little or no effect on the critical micelle concentration. Since the choleretic properties of bile salts are thought to be directly proportional to their osmotic activities, one might suspect that deoxycholic acid would be the least choleretic, chenodeoxycholic slightly more choleretic and cholic much more choleretic, with little difference between the conjugated and unconjugated forms. However, in the present study, cholic, chenodeoxycholic and taurocholic acid produced similar increases in bile flow (450–700 μl/kg) after an equimolar dose (55 μM/kg). Except for the conjugation of deoxycholic acid with taurine, conjugation of these bile acids with glycine or taurine always decreased the choleretic properties of the bile acids. Therefore, it has been concluded that there is not a good correlation between the in vitro osmotic properties of bile acids and their ability to increase bile flow.     

天然牛黄与培育牛黄的扫描电镜观察

天然牛黄(natural ox gallstone)为牛的胆结石,由于产量极少,供求困难,国内于1956年参照天然牛黄的主要成分制成人工牛黄(artificial ox gallstone),作为天然牛黄的代用品。从1974年以来,很多省区如吉林、广东、内蒙古等都先后进行了人工培育牛黄的试验,并试验成功培育牛黄(cultured ox gallstone)。其方法是用手术在牛胆囊内植入异物为核,促使胆汁成分的沉积与结石的形成,经过一段时间后,将异物取出,上面的沉积物即为培育牛黄。本文目的是探讨天然牛黄与培育牛黄的结构特征。     

Inhibiting uptake activity of organic anion transporter 2 by bile acids

Bile acids, a series of amphiphilic molecules, can interact with several drug transporters and impact drug ADME. Organic anion transporter 2 (OAT2) is exclusively expressed in the liver and kidney. However, the interaction between bile acids and hOAT2 is unelucidated. In this study, we observed that chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid, glycoursodeoxycholic acid (GUDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid, tauroursodeoxycholic acid could all inhibit uptake activity of hOAT2 while glycocholic acid (GCA) and cholic acid could not. Among them, TCDCA was the strongest inhibitor with IC₅₀ value of 23.01 ± 3.45 μM and GCDCA was the second with IC₅₀ value of 54.26 ± 5.47 μM. Meanwhile, GCA, GUDCA, TCA and TCDCA were identified as substrates of hOAT2. We further found that bile acid mixture (BA mix) could inhibit hOAT2-mediated uptake of cGMP, 5-fluorouracil, irinotecan and paclitaxel. BA mix could reduce the toxicity of paclitaxel to MDCK-hOAT2 cells. In addition, the uptake activity of three SNPs of hOAT2 (C329T, G571A, and G1514A) was all reduced. In conclusion, this study revealed bile acids could interact with hOAT2, providing new insight into the alteration of drug ADME and therapeutic effect mediated by hOAT2.     

Genotoxicity of dicrotophos, an organophosphorous pesticide, assessed with different assays in vitro

Dicrotophos is a systemic insecticide with a wide range of applications. We investigated the genotoxicity of dicrotophos using the Ames test, the chromosome aberration test in CHO-K1 cells, and the comet assay in the Hep G2 cells, while this chemicals' toxicity to both the cell lines was evaluated with the MTT assay. Results showed that dicrotophos did not show any cytotoxicity to CHO-K1 cells, whereas it was cytotoxic to HepG2 cells incubated for 24 h but not for 2 h. For genotoxicity of dicrotophos, a significant change in the numbers of bacterial reveratnts using Salmomella typhimurium TA97a, TA98, TA100, TA102, and TA1535 as the tester strains, an increase in the frequencies of chromosome aberration in CHO-K1 cells, and an induced DNA damage in HepG2 cells were observed, indicating that dicrotophos was genotoxic in these three performed assays. From this study, we provide further evidence towards of genotoxic effects of dicrotophos.     

Involvement of the multidrug resistance P-glycoprotein in acetaminophen-induced toxicity in hepatoma-derived HepG2 and Hep3B cells

Acetaminophen overdose causes severe hepatic failure. Although the mechanisms of acetaminophen hepatotoxicity have been well investigated, little is known about the involvement of the P-glycoprotein in acetaminophen transport and toxicity. P-Glycoprotein is a membrane efflux pump, playing a significant role in regulating absorption, excretion, and tissue distribution of many drugs. To evaluate the contribution of P-glycoprotein transporter in the course of acetaminophen-induced toxicity, HepG2 and Hep3B cells with different P-glycoprotein expression and activity, were treated by acetaminophen (1-10 mM) for different time periods, with or without the P-glycoprotein inhibitor verapamil. P-Glycoprotein activity was determined by rhodamine 123 efflux assay and western blot analysis. To assess the acetaminophen-induced toxicity and effect of verapamil, we investigated cellular redox status, phosphatidylserine externalization, nuclear fragmentation and ultrastructural changes. Verapamil markedly enhanced acetaminophen-induced oxidative damage and cell death. Moreover, verapamil revealed acetaminophen toxicity even at subtoxic levels. High acetaminophen concentrations increased P-glycoprotein activity and content in both HepG2 and Hep3B cells. These observations suggest the involvement of P-glycoprotein in acetaminophen transport. Notwithstanding the differences of the investigated hepatoma cell lines in P-glycoprotein function, acetaminophen-induced toxicity was similar, possibly due to different functions of drug-metabolizing systems. We conclude that acetaminophen is a P-glycoprotein substrate and P-glycoprotein is involved in acetaminophen transport and toxicity in HepG2 and Hep3B cells. This study establishes the fact that acetaminophen can modulate P-glycoprotein in tumour cells, suggesting that its routine use in cancer patients in combination with anticancer drugs, may influence the result of chemotherapy.     

Altered bile acid metabolism in alloxan diabetic rats

Changes of cholesterol, phospholipid, triglyceride or bile acid levels in serum liver, bile and feces after the treatment with alloxan were examined in Wistar strain male rats. Serum cholesterol, phospholipid and triglyceride levels and liver cholesterol level markedly increased but liver phospholipid and triglyceride levels remained unchanged. The lipid levels in serum very low density and low density lipoproteins were elevated but those in high density lipoprotein were not. Bile flow was not changed but biliary secretion of cholesterol, phospholipid and bile acids markedly increased. Among the biliary bile acid components, cholic acid markedly increased but the amount of chenodeoxycholic acid was similar to that of normal rats. Fecal excretion of deoxycholic acid increased but that of lithocholic and hyodeoxycholic acids decreased, and alpha, beta- and omega-muricholic acids did not change, thus, the total amount of fecal bile acids remained unchanged. Hepatic cholesterol synthesis was markedly depressed, while cholesterol 7 alpha-hydroxylase activity did not change and cytochrome P-450 content was elevated by about 40%. From such evidence, it was apparent that synthesis of cholic acid increased while that of chenodeoxycholic acid decreased and the total amount of bile acids synthesized did not change in the diabetic rats. Furthermore, marked increase of the pool size of cholic acid and hepatic secretion of cholic acid stimulated the absorption of lipids and produced a hyperlipidemia in the diabetic rats.     

Correlation of biliary cholesterol, phospholipids and bile acid compositions and the development of cholesterol cholelithiasis in mice

A study was attempted to establish a screening method for detecting cholelitholytic ingredients from a wide variety of natural substances. Although mice were selected as a suitable pathological model of cholelithiasis to detect a small amount of the ingredients, all the conventional lithogenic diets caused unfavorable influence on the animals. Therefore, as the first step we formulated a new lithogenic diet consisting of butter, cholesterol, cholic acid, etc, which was adequate for mice. Subsequently, the pathological characteristics and persistence of cholelithiasis were examined in the animals; the changes in bile compositions including free and conjugated bile acids, cholesterol and phospholipids were observed before and at the onset of cholelithiasis. Following confirmation of the stone formation, a normal diet was substituted for the lithogenic diet to likewise assess the bile compositions 4 and 6 weeks later. An increasing tendency for deoxycholic acid, disappearance of chenodeoxycholic acid and decrease in ursodeoxycholic acid were seen under the condition of cholelithiasis. In addition, the cholic acid-glycine conjugate which should not exist in the normal state and the increase in free and tauring-conjugated cholic acid were noticed. The biliary cholesterol level in treated mice increased to about 4 times higher than that in untreated mice, while the biliary phospholipids and total bile acids levels increased to only about 1.5 and about 2 times the control levels, respectively. The incidence of stone formation rose sharply at an experimental period between 2 and 3 weeks after starting the lithogenic diet. Gallstones die not disappear even at the 6th week after substituting a normal diet for the lithogenic one. However, the cholic acid-glycine conjugate disappeared, and deoxycholic acid as well as chenodeoxycholic acid and ursodeoxycholic acid tended to recover to the normal levels in the bile.     

高效液相多通道同时测定藏药珍珠丸系列品种中名贵药味所含的6种组分

目的：建立藏药珍珠丸系列品种（二十五味珍珠丸、七十味珍珠丸）中名贵药味西红花和牛黄所含6种组分（西红花苷-Ⅰ,西红花苷-Ⅱ、牛磺胆酸钠、胆酸、猪去氧胆酸、去氧胆酸）含量的HPLC-TUV-ELSD同时测定方法,并对不同企业的样品进行测定和分析。方法：采用Zorbax SB-C₁₈色谱柱（4.6 mm×250 mm,5 μm）,流动相A为乙腈,B为1%甲酸,梯度洗脱程序为0～60 min,18%A～40%A,流速为1.0 mL·min^-1,柱温30℃, TUV检测波长为440 nm,ELSD漂移管温度80℃,雾化气压力为30 psi。结果：在选定的条件下,西红花苷-Ⅰ,西红花苷-Ⅱ、牛磺胆酸钠、胆酸、猪去氧胆酸、去氧胆酸6种组分分离良好,线性范围合理（r>0.9932）,加样回收率范围为97.5%～102.1%（n=6）,LOD分别为0.0001,0.0002,0.005,0.002,0.003,0.002 g·L^-1。结论：所建立的方法简便、快速、准确,能用于同时检测珍珠丸系列样品中6种组分的含量,为方中名贵药味的质量控制提供技术参考,并对市售样品进行质量分析。     

Transformation of hamster embryo cells by neutral sterols and bile acids

Toxicity and cell transformation of Syrian hamster embryo cells in culture by certain neutral sterols and bile acids show interesting trends related to their structures: cholesterol-α-epoxide and cholestan-3β,5α,6β-triol were more toxic and induced transformation to these cells, whereas their metabolic precursor, cholesterol, was inactive. The secondary bile acids, lithocholic and deoxycholic acids, were more toxic than their primary bile acid precursors, cholic and chenodeoxycholic acids and transformed the cells. These data suggest that mammalian cell transformation is a useful short-term assay to measure the potential toxicity and carcinogenicity of steroid derivatives.     

Olefine der Cholanreihe aus Desoxycholsäure und Cholsäure

Olefins of the Cholane Series from Deoxycholic and Cholic Acids On partial dehydration deoxycholic acid yielded two new olefins. By the same procedure five new olefins were obtained from cholic acid.     

Differential pulmonary in vitro toxicity of two small-sized polyvinylpyrrolidone-coated silver nanoparticles

Silver nanoparticles (AgNP), with their important properties, are being used in a range of sectors from industry to medicine, leading to increased human exposure. Hence, their toxicity potential needs to be comprehensively evaluated. It was postulated that within small-sized (≤20 nm) polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNP), minor size differences may significantly induce different toxicity profiles and involve varying cellular pathways. Therefore, the aim of this study was to examine the influence of differing size AgNP with 10 nm (AgNP10) and 20 nm (AgNP20) (up to 100 µg/ml), as well as to ionic silver as AgNO₃ for 24 and 48 h, using the human lung cell line A549. The effects on cell viability, proliferation, apoptosis, DNA damage and cell cycle dynamics were assessed. Results for both time periods showed that for low concentrations (<5 µg/ml), AgNP20 were more cytotoxic than AgNP10, however, at higher doses, AgNP10 exhibited higher toxicity. For concentrations >50 µg/ml, AgNP10 induced severe DNA damage (comet class 3–4), cell cycle arrest at G₂ phase and late-stage apoptosis, while AgNP20 induced cell cycle arrest at S phase and an increase in the percentage sub-G_1, which did not recover after 48 h, and late-stage apoptosis/necrosis. In longer-term exposures, the greater impairment in colony formation due to AgNP exposure than to silver ion supports that nanotoxicity is not exclusively due to the released ion. Data suggest that toxicity mediated by small AgNP (≤20 nm) in lung cells is not only dependent on the level of particle internalization, but also on AgNP size and concentration, which may involve varying pathways as targets.