Study of the potential toxicity of commercial crispy breads by evaluation of bioaccessibility and bioavailability of minor Fusarium mycotoxins

Enniatins (ENs) are bioactive compounds produced by the secondary metabolism of several Fusarium strains and known to have several biological activities, such as acting as enzyme inhibitors, antifungal and antibacterial agents, and immunomodulatory substances. This study has investigated the ENs bioaccessibility, spiked in commercial wheat crispy bread at 1.5 and 3.0μmol/g concentrations, their transepithelial transport and bioavailability using Caco-2 cells as a model of the human intestinal epithelium. The content (%) of the four ENs contained in the gastric fluid has resulted variable from 69% to 91%, considering the two concentrations assayed. The mean bioaccessibility data for the compounds studied, resulted of 80%. The compounds that evidenced the highest absorption, using the in vitro model which simulated the transepithelial transport, were the EN A (70.8±1.3% of absorption) and A(1) (73.8±0.9%) at 1.5 and 3.0μmol/g concentrations, respectively. The compound with the lowest transport value (50.7±1.3%) was the EN A at 3.0μmol/g concentration. The bioavailability data evidenced by the other ENs employed ranged from 55.2±1.1% to 66.1±1.0%.     

Different transport mechanisms for cadmium and mercury in Caco-2 cells: inhibition of Cd uptake by Hg without evidence for reciprocal effects

Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203Hg(II) and 109Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K(i) for Hg of 9.3 +/- 1.2 microM) without reciprocal effects. 109cadmium uptake was highly sensitive to temperature and a significant fraction of accumulation (12%) was EDTA extractable. 203Hg uptake remained insensitive to temperature or the EDTA washing procedure. However, the uptake of both tracers was half-decreased when an excess of the respective unlabeled metal was added in the stop solution, suggesting an exchange mechanism for adsorption. Cell pretreatment with N-ethylmaleimide (NEM) led to a 30% decrease or a 73% increase in the 3-min specific transport of 109Cd when NEM was still present in or removed from the uptake medium, respectively. NEM had no effect on 203Hg uptake. Overall our results suggest the involvement of a saturable specific mechanism for Cd, which is highly sensitive to inhibition by Hg and NEM under some conditions, and a nonspecific passive diffusion for Hg. The Hg- or NEM-induced inhibition of Cd uptake likely involves a thiol-mediated reaction, but our results suggest that NEM pretreatment may activate other cellular mechanisms leading to a stimulatory effect.     

The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim： Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. There is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux （ABCB1, ABCG2 and ABCC2） and uptake （SLCO1B1, SLCO2B1 and SLC22A1） drug transporters in Caco-2 and HepG2 cells were investigated.
Methods： Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results： Differences in mRNA basal levels of the transporters were as follows： ABCC2〉ABCG2〉ABCB1〉SLCO1B1〉〉〉SLC22A1〉SLCO2B1 for HepG2 ceils, and SLCO2B1〉〉ABCC2〉ABCB1〉ABCG2〉〉〉SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABOG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1, 10 and 20 pmol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRNA levels after 12 or 24 h treatment.
Conclusion： These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may con- tribute to the variability in oral disposition of statins.     

HPLC-MS法测定关附甲素及其在Caco-2细胞上的摄取特性

目的建立关附甲素的LC-MS测定法,研究其在Ca-co-2细胞上的摄取特性。方法采用ShimadzuVP–ODS,C8柱,(250mm×2.1 mm,5μm),流动相:乙腈∶水=30∶70(V/V),用0.2%冰乙酸溶液,加入二乙胺适量调pH至4.0左右,流速:0.2 ml.min-1,电喷雾离子化(ES1)方式,采用选择性离子检测法,检测离子为正离子,关附甲素的选择性离子:[M+H],m/z 430。考察温度、药物浓度、时间和pH值对关附甲素在Caco-2细胞上摄取的影响。结果关附甲素在0.2～50.0μmol.L-1的范围内呈良好的线性关系(r=0.9988),最低定量限0.2μmol.L-1。关附甲素在Caeo-2细胞上的摄取具有一定的浓度、时间和pH值依赖性,其摄取表现为被动扩散。结论该法灵敏、简单,专属性强,关附甲素在Caco-2细胞上具有良好的摄取。     

Effect of thiamine on H-MPP uptake by Caco-2 cells

Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP⁺ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP⁺ intestinal apical uptake and ecto-ALP activity by thiamine (T⁺) and thiamine pyrophosphate (TPP, a T⁺ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-³H]-4-phenylpyridinium acetate (³H-MPP⁺) uptake were evaluated in intact Caco-2 cells. T⁺ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease ³H-MPP⁺ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and ³H-MPP⁺ uptake were concentration-dependent. The results suggest that the effect of T⁺ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T⁺ may occur through hEMT, in Caco-2 cells.     

儿茶素对Caco-2细胞胆固醇摄取的影响及机制研究

目的： 研究儿茶素（Catechin，Cat）对Caco-2细胞胆固醇摄取的影响，以及可能的作用机制。方法： 利用胆固醇：甲基β环糊精复合物（Chol：MβCD）建立Caco-2细胞脂质蓄积模型。不同浓度的儿茶素（20、40、60 μmol·L^-1）处理细胞24 h，结合油红O染色法观察细胞内的脂质蓄积，酶法测定细胞内胆固醇含量及分布，qRT-PCR及Westernblot检测胆固醇代谢相关基因NPC1L1和SREBP-2的表达。结果： 与空白组相比，Chol：MβCD处理组细胞内红色脂滴颗粒以及胆固醇含量明显增加。20~60 μmol·L^-1儿茶素不仅可以不同程度地减少细胞内红染脂滴的形成，而且可以显著降低细胞内总胆固醇和游离胆固醇的含量，以及胆固醇酯在总胆固醇中的比例。此外，儿茶素可以剂量依赖性地降低胆固醇代谢相关基因NPC1L1及SREBP-2的mRNA和蛋白表达。其中以60 μmol·L^-1 Cat处理组的作用最为显著（P<0.01）。结论： 儿茶素可能通过下调胆固醇代谢相关基因NPC1L1和SREBP-2的表达，进而减少Caco-2细胞摄取胆固醇及蓄积。     

蜕皮甾酮在Caco-2细胞模型中的摄取和跨膜转运研究

目的以Caco-2细胞单层模型,首次研究了蜕皮甾酮的口服吸收与转运特性。方法采用普通Caco-2细胞模型、表达活性CYP3A4酶的Caco-2细胞模型,分别从AP→BL方向和BL→AP方向研究蜕皮甾酮的摄取和跨膜转运规律。结果蜕皮甾酮在2种模型中的表观渗透系数(Papp)在0.1×10-6～1×10-6cm.s-1之间,药物吸收情况为1%～10%;在4 h的实验过程中,4种浓度蜕皮甾酮的ER值均小于1.5。结论研究表明,蜕皮甾酮主要以被动扩散的方式被细胞摄取和转运,其跨膜转运特性未受到CYP3A4-介导机制的影响。     

神经酰胺对人结肠癌Caco-2细胞中谷胱甘肽硫转移酶A1的影响

谷胱甘肽S-转移酶(GSTs)是一组多功能Ⅱ相药物代谢酶,能够清除外源性的亲电子化合物,包括致癌物、氧化应激产物和化疗药物等。己有研究表明,GSTs的过表达与肿瘤细胞化疗耐药性的产生存在着密切的关系,某些肿瘤耐药细胞株内GSTs含量要比敏感株高出许多倍,肿瘤细胞可通过表达GSTs而保护自身免受化疗药物的损害,是机体防御机制的     

Ochratoxin A secretion by ATP-dependent membrane transporters in Caco-2 cells

The ATP-dependent membrane transporters, P-gp, MRP2 and BCRP, localized in the luminal membranes of the intestines, liver and kidney, counteract absorption and increase excretion of xenobiotics and drugs. Previously, it has been suggested that the mycotoxin ochratoxin A (OTA) is a substrate for ATP-dependent transporters, and hence the absorption and secretion of OTA in the Caco-2 cell model was investigated. To this end, Caco-2 cells were cultured as confluent monolayers in bicameral inserts and the transepithelial transport of the mycotoxin was assessed. Caco-2 cells secreted OTA to the luminal side in a concentration-dependent manner. This secretory permeability was higher than the absorptive permeability, while the absorptive permeability remained constant for all OTA concentrations tested. The secretion decreased and absorption increased in the presence of the MRP-inhibitor MK571, the P-gp and BCRP inhibitor GF120918, and the BCRP-inhibitor Ko143, suggesting that the secretion of OTA is mediated by MRP2 and BCRP. Cyclosporine A also decreased the secretory permeability, but did not affect absorptive permeability, while PSC833 did neither change absorption nor secretion of OTA. Hence it can be suggested that OTA is a substrate for MRP2 as well as BCRP. These findings are of interest in evaluating mycotoxin absorption after oral ingestion, tissue distribution and particularly excretion pathways, including renal, biliary and mammary gland excretion.     

枸杞多糖对Caco-2细胞P-糖蛋白作用的影响

目的 研究枸杞多糖(LBP)对Caco-2细胞P-糖蛋白(P-gp)功能和表达的影响.方法 采用MTT法确定药物实验剂量;采用Rh-123摄取法评价P-gp的功能;利用流式细胞术与免疫荧光抗体检测P-gp的表达;采用经典的Caco-2细胞单层模型,研究P-gp外向转运这一动态过程受枸杞多糖作用后的变化.结果 由MTT法得到枸杞多糖＜100μg·mL-1时,细胞的存活率＞90％;枸杞多糖使细胞内Rh-123累积增加,对功能表现为抑制作用;高浓度组(100μg·mL-1)枸杞多糖对P-gp表达有诱导作用;高浓度组(100μg ·mL-1)枸杞多糖使Caco-2细胞单层模型外排率(ER)降低,对P-gp有抑制作用.结论 枸杞多糖对P-gp功能表现为抑制作用,而对P-gp表达表现为诱导作用.     

顺式-和反式-阿霍烯在Caco-2细胞模型中的体外摄取、转运和外排特性

研究顺式-阿霍烯(Z-Ajo)和反式-阿霍烯(E-Ajo)的肠细胞摄取、转运和外排特性。采用体外培养的人结肠Caco-2细胞单层模型评价，应用高效液相色谱法测定Z-Ajo和E-Ajo的含量。结果表明，仅能在Caco-2细胞单层的顶侧检测到Z-Ajo或E-Ajo；阿霍烯在Caco-2细胞中的代谢可被抗氧化剂维生素C、细胞色素P450药物代谢酶3A亚型抑制剂甲吡酮和ATP抑制剂叠氮化钠所抑制。Z-Ajo和E-Ajo皆不能透过Caco-2单层细胞而被迅速代谢，其代谢与CYP450药物代谢酶有关。     

Caco-2细胞模型在药物研究中的应用

Caco2细胞模型广泛用于药物的吸收、代谢以及毒性研究,Caco2细胞模型作为药物吸收研究的一种快速筛选工具,在抗癌药物、无机药物、中药的研究方面得到了广泛的应用,成为药物研究的重要手段。     

Intestinal transport of methylmercury and inorganic mercury in various models of Caco-2 and HT29-MTX cells

Food is the main pathway of exposure to mercury for most of the population. In food, mercury is generally present as inorganic mercury [Hg(II)] or methylmercury [MeHg]. Both chemical forms have some degree of toxicity, especially MeHg, which is considered a powerful neurotoxicant during development and is classified as a possible human carcinogen. Since the main exposure pathway is oral, gastrointestinal absorption is a decisive step in the process by which mercury reaches the systemic circulation. However, there are few studies that characterize this absorption process.     

灵芝多糖肽对Caco-2细胞上P糖蛋白功能、表达的影响

目的 研究灵芝多糖肽(ganoderma lucidum polysaccharides peptide,GLPP)对Caco-2细胞P糖蛋白(P-gp)功能和表达的影响.方法 用流式细胞仪测定Caco-2细胞中P-gp底物罗丹明123和抗体的荧光强度,分析药物对P-gp功能和表达的影响.双向跨膜转运实验考查GLPP对罗丹明123转运的影响.结果 10、20、50、100μg·mL-1的GLPP均抑制了Rh-123的外排,其外排分别降低了8％、16％、19％、33％,具有浓度依赖性.高、中浓度的GLPP(50、100 μg·mL-1)对Rh-123的双向转运有一定的抑制,外排率(ER)较阴性对照组降低了15％、18％.细胞与药物作用72 h后,细胞抗体荧光强度降低,P-gp的表达降低.结论 GLPP对P-gp的活性有一定的抑制作用,能在较短的时间内增加细胞内Rh-123的蓄积,抑制Rh-123的双向转运.长期使用GLPP可抑制P-gp的表达.     

大鼠小肠原位灌流与Caco-2细胞法研究柠檬苦素吸收机制

柠檬苦素广泛存在于柑橘类水果中, 具有抗菌、抗病毒、镇痛、抗炎和抗癌等活性, 但其口服生物利用度较低。本文意在研究柠檬苦素在肠道内的吸收机制, 为其今后的研究奠定基础。实验通过大鼠原位肠灌流和体外Caco-2细胞法进行。大鼠原位肠灌流结果显示, 柠檬苦素可能通过肠道促进扩散机制吸收, 吸收差且在全肠段都有吸收, 没有部位选择性。Caco-2细胞实验结果显示, 维拉帕米和酮康唑能显著提高柠檬苦素的吸收, 而丙磺舒的影响不明显。柠檬苦素吸收较低和生物利用度较差, 可能是P-gp外排以及CYP3A4代谢共同参与的结果。柠檬苦素的肠吸收机制研究将为其剂型设计和临床应用提供重要的参考。     

9-硝基喜树碱在Caco-2细胞模型中的体外摄取、转运及外排动力学

目的研究9-硝基喜树碱(9-NC)的细胞摄取、转运及外排特性。方法一种体外培养的人小肠上皮细胞模型Caco-2应用于9-NC的小肠上皮细胞的摄取、跨膜转运及外排动力学研究。评价了时间、温度、pH,P-糖蛋白(P-glycoprotein, P-gp)抑制剂对细胞摄取的影响。采用HPLC测定药物含量。结果9-硝基喜树碱以被动扩散为主要方式被细胞摄取和转运。药物的摄取与时间呈正相关，与温度、pH呈负相关。P-gp抑制剂环孢菌素和维拉帕米增加9-NC细胞摄取(P<0.05)。药物从Basolateral(B,基底面)到Apical(A,肠腔面)的渗透系数P_app大于A到B(2.6-6.9倍)。9-NC外排符合二级外排动力学过程，A侧m₀［(148.0±2.2) pmol·cm^-2］和外排速率(41.1 pmol·cm²·min^-1)高于B侧的m₀［(121±7) pmol·cm^-2(P<0.05)和外排速率(29.2 pmol·cm²·min^-1)(P<0.01)。结论 9-NC是以被动扩散方式为主要方式被小肠上皮细胞摄取和转运，并受到P-糖蛋白强烈的外排作用。     

左旋紫草素肠道转运及Caco-2细胞转运特性

目的研究左旋紫草素肠道及Caco-2细胞转运特征及其机制。方法应用翻转肠囊法和Caco-2细胞模型考察时间、浓度对左旋紫草素转运吸收特性的影响,应用P-糖蛋白抑制剂维拉帕米对左旋紫草素转运吸收机制进行研究,采用HPLC法测定左旋紫草素的浓度,计算其表观渗透系数(Papp)。结果在Caco-2细胞模型,随浓度增加和时间延长,左旋紫草素的累积转运量逐渐增加;加用维拉帕米后,使AP侧到BL侧的表观渗透系数Papp(AP→BL)显著增加,而从BL侧到AP侧的表观渗透系数Papp(BL→AP)显著降低。在翻转肠囊模型,100μmol·L-1左旋紫草素中加入维拉帕米后Papp显著增加。结论左旋紫草素的转运存在被动转运和主动转运2种形式,P糖蛋白参与主动转运过程;该药经肠道吸收中等,加入维拉帕米可能促进吸收。     

利用人源Caco-2细胞单层模型研究罂粟碱、N-甲基四氢罂粟碱和头花千金藤碱在人肠道的吸收

研究罂粟碱(papaverine，PAP)、N-甲基四氢罂粟碱(laudanosine，LAU)和头花千金藤碱(cepharanthine，CEP)在人小肠的吸收。利用人源结肠腺癌细胞系Caco-2细胞单层模型研究PAP、LAU和CEP由绒毛面(AP侧)到基底面(BL侧)、BL侧到AP侧两个方向的转运过程。应用HPLC-UV对上述3个生物碱进行定量分析，计算转运参数和表观渗透系数，并与易吸收性对照药普萘洛尔和难吸收性对照药阿替洛尔进行比较。PAP、LAU和CEP由AP侧到BL侧的表观渗透系数(P_app)分别为(3.524±0.223)×10^-5、(2.821±0.050)×10^-5和(6.524±0.052)×10^-5 cm·s^-1；由BL侧到AP侧的P_app分别为(5.095±0.508)×10^-5、(2.646±0.146)×10^-5和(5.495±0.036)×10^-5 cm·s^-1，与在Caco-2细胞单层模型上呈良好吸收的阳性对照药普萘洛尔基本一致。PAP、LAU和CEP的P_{app A→B}/P_{app B→A}分别为0.69、1.07和1.19；PAP外流是摄取的1.45倍。PAP、LAU和CEP可以通过小肠上皮细胞被动吸收进入体内，属于良好吸收的药物。在三者的吸收转运过程中，油/水分配系数起着关键性的作用。PAP在Caco-2细胞单层模型中的转运可能存在外流机制。     

盐酸赛庚啶在Caco-2细胞中吸收转运的研究

目的 研究盐酸赛庚啶在Caco-2细胞模型中的吸收机制。方法 建立Caco-2细胞体外吸收模型,通过测定荧光黄透过率、安替比林和普萘洛尔的渗透系数、地高辛的外排率验证Caco-2细胞吸收模型的成功建立。采用LC-MS/MS法测定盐酸赛庚啶在膜两侧的浓度,计算渗透系数和外排率。结果 Caco-2模型建立成功,采用LC-MS/MS法可以准确测定缓冲体系中盐酸赛庚啶的浓度。结论 盐酸赛庚啶的渗透系数随药物浓度的变化而变化,且与转运方向有关,可能为P-gp和BCRP的底物。