A possible reason for the phalloidin tolerance of hepatoma cells

Summary In contrast to normal liver cells, AS-30D hepatoma cells are insensitive to phalloidin. The lack of the typical phalloidin response in the latter cells is not due to a deficiency of contractile proteins. Actin prepared from hepatoma cells is able to form filamentous structures and is stabilized in a manner similar to muscle actin.Isolated liver cells were exposed to a medium containing phalloidin and removed after 20 min by centrifugation. The supernatant was inculated again with fresh cells. The procedure was repeated four times. The phalloidin response decreased to about 19% of the control because of the uptake of phalloidin during each incubation. When the same procedure was carried out with AS-30D hepatoma cells, and aliquots of the supernatants were tested with hepatocytes no marked decrease of the phalloidin response was seen. This indicates that hepatoma cells do not consume the toxin as do normal liver cells.This work was supported by the Deutsche Forschungsgemeinschaft     

可回复性肝细胞永生化载体pCTGTKIox的构建、鉴定、转染与表达

目的构建可回复性永生化逆转录病毒载体，为肝细胞可回复性永生化奠定基础。方法扩增加强型绿色荧光蛋白（EGFP）及胸腺激酶（TK）并采用重叠延伸拼接法（SOE）连接EGFP和TK，将EGFP-TK（融合基因亚克隆至pBABE-puro-lox，构建成新载体pBGTKlox，再将永生化基因SV40T连接至pBGTKlox，构成新载体pBTGTKlox，最后将雌激素受体与重组酶融合基因（Cm-ER）连接至pBTGTKlox，构成新载体pCTGTKlox。将pCTGTKlox和pPDF15质粒共转染包装细胞PT67，并在荧光显微镜下观察绿色荧光蛋白的表达。免疫组化染色检测SV40T基因的表达。结果成功构建包含EGFP-TK、SV40T、Cre-ER及重组序列loxP的新载体pCTGTKlox，经酶切鉴定证实为重组阳性体，经测序验证无核苷酸突变。pCTGTKlox转染PT67细胞24h后，荧光显微镜下可见散在多处绿色荧光。免疫组化染色显示细胞胞核呈阳性染色。结论成功构建并表达可回复性永生化逆转录病毒载体pCTGTKlox，为细胞的可回复性永生化提供了一种良好的新型载体。     

Deformation of isolated rat hepatocytes by a peptide hepatotoxin from the blue-green alga Microcystis aeruginosa

Summary The effect of the peptide hepatotoxin from the bloom-forming blue-green alga Microcystis aeruginosa was investigated on isolated rat hepatocytes. When toxin was added to hepatocyte suspensions it produced deformation of the cells, as shown by scanning electron microscopy. This was apparent within 5 min of addition of toxin to the cells and the response was dose dependent: 30 ng of toxin was sufficient to cause deformation in 58±9% of 1.4×10⁶ hepatocytes/ml of incubation. The deformation did not lead to cell death as measured by Trypan blue uptake within 120 min.Deoxycholate, cholate, bromosulphophthalein, and rifampicin were found to prevent the deformation of hepatocytes by Microcystis aeruginosa toxin in a dose dependent manner, analogous to the effect of these agents on the response of hepatocytes to added phalloidin. This suggests that Microcystis aeruginosa toxin is transported into hepatocytes in the same way as phalloidin; namely sharing a transport system for bile acids on the hepatocyte plasma membrane.Part of this publication was presented to the Australian Biochemical Society Conference in May, 1981     

肝细胞刺激因子对环磷酰胺致小鼠肝细胞损伤的保护作用

目的 研究环磷酰胺(CTX)对肝细胞的损伤及肝细胞刺激因子(HSS)对该损伤的保护作用。方法 应用原代小鼠肝细胞混悬培养，检测肝细胞培养液中乳酸脱氢酶(LDH)活性和肝细胞谷胱甘肽(GSH)、丙二醛(MDA)含量变化。结果 CTX可使LDH漏出增多，同时肝细胞GSH含量减少，MDA含量增加，HSS可部分逆转上述变化。结论 CTX可致肝细胞损伤，HSS降低肝细胞MDA含量可能是其保肝机制之一。     

肝星状细胞和肝细胞合成及分泌胶原的比较研究

目的 为了明确肝细胞(hepatocyte，HC)和星状细胞(hepatic stellate cell,HSC)在肝纤维化胶原合成和分泌中的作用。方法 本文在分离培养大鼠HC和SHC的基础上，用H—L—胰氨酸掺入法和胶原酶消化法分析体外培养的HC和HSC胶原合成和分泌功能。结果 发现HC和HSC合成胶原的能力相似(P＞0．05)，而HSC分泌胶原能力较强(P＜0.05—0．01)。结论 HSC在肝脏胶原合成和分泌中可能起主要作用，但HC的作用也不容忽视。     

Hepatotoxicity of diisopropyl ester of malonic acid and chloromalonic acids, disinfection by-products of the fungicide isoprothiolane

The fungicide isoprothiolane (diisopropyl 1,3-dithiolane-2-ylidenemalonate) decomposes to the diisopropyl esters of malonic acid (DM), chloromalonic acid (DCM) and dichloromalonic acid (DDCM) upon aqueous chlorination. In this study, the cytotoxicity of these compounds was examined using rat hepatocytes cultured on Matrigel. DCM and DDCM caused hepatocellular death at concentrations >0.5 mM, while DM had no effect on the cell viability even at the maximum concentration examined (4 mM). Significant lipid peroxidation, measured as 2-thiobarbituric acid reactive substances, was observed in both DCM- and DDCM-treated hepatocyte cultures, and was significantly enhanced by pretreatment with 0.1 mM bis( p-nitrophenyl)phosphate (BNPP), a carboxylesterase inhibitor. When both BNPP and SKF-525A, a cytochrome P450 inhibitor, were present in the medium, DCM-induced cytotoxicity and lipid peroxidation were significantly suppressed compared to cultures with BNPP-treatment alone. By contrast, the DDCM-induced cytotoxicity was not affected by the combined pretreatment of SKF-525A and BNPP. These results indicate that DCM is metabolically activated by cytochrome P450 in an ester form, while DDCM is activated by a mechanism other than one involving cytochrome P450. To further elucidate the cytochrome P450 isozyme involved in the metabolic activation of DCM, microsomal lipid peroxidation was studied in vitro using microsomes from rats treated with β-naphthoflavone, musk xylene, phenobarbital, pyrazole, or dexamethasone. Among these preparations, the microsomes from dexamethasone-treated rats showed the most extensive lipid peroxidation in the presence of DCM, and the lipid peroxidation was enhanced by BNPP as observed in hepatocyte cultures. These findings suggest the possible involvement of cytochrome P450 3A in the metabolic activation of DCM. Received: 12 February 1997 / Accepted: 10 April 1997     

HYPOTHERMIC ISCHAEMIA OF THE LIVER: A RE-PERFUSION PHENOMENON

Background : The effects of hypothermic injury to the liver were investigated on an isolated perfusion circuit by comparing porcine livers with varying degrees of preservation injury. Methods : A group of unstored livers (n= 5) were compared to livers stored in University of Wisconsin (UW) solution for 18 h (n= 5), and a group of livers stored in Hartmann's solution for 18 h (n= 5). Results : We observed that the degree of platelet sequestration was directly related to the severity of the preservation injury. After 2 h of isolated liver perfusion, the perfusate platelet count fell from 148 ± 14 × 10⁹/L to 84 ± 13 × 10⁹/L for control livers. In comparison for livers stored in UW solution, the platelet count fell from 173 ± 43 × 10⁹/L to 61 ± 14 × 10⁹/L, representing a 64.8% fall, while for those stored in Hartmann's solution, an even more profound fall from 152 ± 36 × 10⁹/L to 19 ± 9 × 10⁹/L (87.5% fall) was observed. The difference between the UW-stored and Hartmann's-stored livers was significant (P < 0.05). However, using this model, the degree of leukocyte sequestration did not differentiate the groups. Both histological and ultrastructural examination of liver biopsies taken immediately following revascularization demonstrated that for mild degrees of preservation injury following hypothermic storage, changes occur to the sinusoidal lining cells well before changes to the parenchymal elements. Conclusions : These findings substantiate the hypothesis that the primary injury associated with hypothermia involves the sinusoidal lining cells (non-parenchymal elements), that it is predominantly a reperfusion phenomenon and that efforts at improving preservation should therefore be targeted primarily at these cells and not the hepatocytes.     

普鲁泊福对原代培养人肝细胞CYP3A4同工酶的影响

目的：观察不同浓度普鲁泊福对原代培养肝细胞中CYP3SA4同工酶活性及蛋白表达的影响，并进一步探讨其临床药理学意义。方法：取肝血管瘤患者的瘤旁正常肝组织标本，胶原酶消化制备原代培养肝细胞，不同浓度普鲁泊福孵育24h后，Nash法测定CYP3A4同工酶比活性，Western印迹法测定CYP3A4蛋白表达，结果：临床剂量（0．01mmol/L)的普鲁泊福即可抑制CYP3A4同工酶的活性，且酶的活性与普鲁泊福的浓度呈负相关，临床剂量的普鲁泊福对该同工酶蛋白的表达并无显著影响，直至浓度为0．5mmol/L时才轻度抑制CYP3A4蛋白表达，结论：麻醉药普鲁泊福对CYP3A4产生明显的抑制作用，可能是一种潜在的P450抑制剂，会导致CYP3A4底物的生物转化速率的下降，其作用主要在表达后水平。     

Glutathione S-transferases in primary cultured rat hepatocytes

The present study is aimed to elucidate the changes in glutathione S-transferase (GST) activity and GST subunit components in primary cultured rat hepatocytes. Enzyme activity was measured with l-chloro-2,4-dinitrobenzene as cosubstrate. The activity decreased at 48 hr, and subsequently increased and returned to levels initially observed at 12 hr by 120 hr. Phenobarbital caused an induction of GST activity in culture at 72 and 168 hr. Immunocytochemical studies were performed using a peroxidase-anti-peroxidase technique with three polyclonal antibodies: anti-Ya, Yb₁ and Yp. With anti-Ya, hepatocytes were persistently positive up to 144 hr in cell culture. With anti-Yb₁, hepatocytes were positive at 24 hr, though positivity then gradually decreased. On the other hand, with anti-Yp, cells were almost negative at 48 hr and became obviously positive at 96 hr. Immunoelectron microscopy with anti-Ybj using the avidin-biotin ferritin method revealed ferritin particles in the ribosomes on endoplasmic reticulum as well as in the free cytoplasmic space. In conclusion, the GST subunit components are in a state of dynamic change in cultured rat hepatocytes, and overall time-dependent increase in the total activity of the enzyme can be accounted for by increased expression of the Yp subunit. Finally, the intracellular localization of Yb₁ subunit was clarified in the present report.