Protective Effect of Dimethyl-4,4'-Dimethoxy-5,6,5',6'-Dimethylene Dioxybiphenyl-2,2'-Dicarboxylate (DDB) against Carcinogen-Induced Rat Liver Nuclear DNA Damage

The protective effect of DDB against carcinogen-induced DNA damage was examined in the present investigation. Preincubation of rat liver nuclei with DDB (1 mmol.L-1) resulted in 60% inhibition of binding of 3H-benzo (a) pyrene to nuclear DNA. Unscheduled DNA synthesis (UDS) induced by aflatoxin BI (10^(-7) mol.L-1) in freshly isolated rat hepatocytes was also inhibited by DDB (10^(-6)-10^(-3)mol.L-1). Oral administration of DDB at 200 mg.kg-1 once daily for 3 d induced a significant increase of liver cytosol glutathione-S-transferase and microsomal UDPG-transferase activity in mice. These results indicate that DDB is able to directly or indirectly antagonize certain carcinogen-induced DNA damages.

Formation of Superoxide Radical and Hydrogen Peroxide Enhanced by Trinitrotoluene in Rat Liver, Brain, Kidney, and Testicle in Vitro and Monkey Liver in Vivo

Trinitrotoluene (TNT) increased the formation of adrenochrome from adrenaline and the formation of formaldehyde from methanol in rat liver mitochondria and microsomes in vitro as well as in monkey liver mitrochondria and microsomes in vivo. The effects were more prominent at higher TNT concentrations. These findings indicate that TNT enhances the production of superoxide radicals (O_2^-) and hydrogen peroxide (H_2O_2). The production of O_2^- was more prominent in systems containing added TNT than in those containing added benzyl viologen. H_2O_2 production by mitochondria was more pronounced in the liver than in other organs, but its production by microsomes was more pronounced in the brain than in other organs. The results suggest that TNT undergoes cycling reduction which produces oxidative stress. 1989 Academic Press, Inc.

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Oxidative Metabolism of Estrone Modified by Genistein and Bisphenol A in Rat Liver Microsomes

Genistein, the main isoflavone from soy, and bisphenol A （BPA）, a food contaminant, are considered ubiquitous xenoestrogens. Here we investigated the influence of genistein and BPA on estrone （El） metabolism in rat liver microsomes. Both substances inhibited the 2-hydroxylation and 16a-hydroxylation of E1, but in different degrees, thereby reducing the 2-OH-E1/16a-OH-E1 ratio,

MECHANISM OF PROMOTING EFFECTS OF RIBOFLAVIN DEFICIENCY ON CARCINOGENESIS OF NITROSAMINES (EFFECTS ON RAT LIVER GLUTATHIONE CONTENT)

The effects of riboflavin deficiency and simultaneously nitrosodimethylamine (NDMA) given by gastric intubation on the hepatic glutathione (GSH) content were examined in rats. On different days of the experiment, hepatic GSH content of the riboflavin deficient rats decreased to 55-61% of the control rats. When NDMA was given 6 mg kg by gastric intubation to riboflavin deficient rats, hepatic GSH content decreased markedly to 39-43% of the control rats. After supplying riboflavin, hepatie GSH content of the deficient rats recovered to the level of the control rats. These results suggest that alterations of rat hepatic GSH content during riboflavin deficiency may imply as one of the promoting effects of riboflavin deficiency on the carcinogenesis of nitrosamines.

A NEW METHOD OF ISOLATING KUPFFER CELLS FROM BIOPSY TISSUE OF RAT LIVER

The isolation of a high yield and purity of Kupffer cells has been reported in detail.1 This paper reports into the research about isolation Kupffer cells from biopsy tissue of liver. This method includes 5 important steps: (1) take fresh liver tissue, and mince with scissors. (2) spin at low speed to wash off red blood cells. (3) digest in collagenase for suitable time. (4) isolate Kupffer cells on a percoll density gradient. (5) cell charaterization was observed by N.S.E stain and peroxidatic activity with lumino-meter measurement and phagocytosis with latex beads.2.3

Iron-Mediated Oxidative DNA Damage Detected by Fluorometric Analysis of DNA Unwinding in Isolated Rat Liver Nuclei

Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.

EXPRESSION OF c-ras-P21 AND GAMMAGLUTA-MYLTRANSFERASE DURING RAT LIVER REGENERATION

c-ras-P21 and Gamma-glutamyltransferase (G-GT) activity and its isoenzymes in rat liver extracts were measured after partial hepatectomy. There were G-GT activity peaks at 12th and 36th hour after operation, while no distinct isoenzyme patterns were found. P21 protein expression occurred between 48 and 96 hours after partial hepatectomy. These results suggest that the temporary increase of G-GT and P21 protein level is involved in the prereplica-tive stage of liver regeneration.

Ursodeoxycholic acid treatment improves hepatocyte ultrastructure in rat liver fibrosis

AIM: To examine the ultrastructural changes after ursodeoxycholic acid (UDCA) treatment in hepatocytes from experimentally induced fibrotic livers.METHODS: Liver fibrosis was induced in male Sprague-Dawley rats with CCl4 for 12 wk, and the rats were divided into two groups. Group I was treated with saline and group Ⅱ with UDCA (25 mg/kg per day) for 4 wk. All the rats were killed at wk 16. Mitochondria, nuclei, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) of hepatocytes were evaluated according to a scoring system.RESULTS: Mitochondria, nuclei, RER and SER injury scores in group Ⅱ were significantly lower than those in groupⅠ(P < 0.001). CONCLUSION: UDCA alleviates hepatocyte organelle injury in CCl4-induced liver fibrosis.

Expression of tissue inhibitor of matrix metalloproteinases-1 during aging in rat liver

AIM: To investigate the expression and role of tissueinhibitor of matrix metalloproteinases-1 (TIMP-1) during natural aging in rat liver and to detect the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9.METHODS: The rats were divided into 3-mo-old group (n = 5), 10-mo-old group (n = 5) and 24-mo-old group(n = 5). Histopathologic changes of liver were observed with HE and Masson stain. The location and protein expressions of TIMP-1 were determined by immunohistochemistry and Westem blot; message RNA (mRNA) levels were measured in livers from rats of various ages by semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR). In addition, the expression of MMP-2 and MMP-9was assessed by RT-PCR and Western blot.RESULTS: Histologic examination showed that the aging liver had excessive fatty degeneration and collagen deposition. Immunohistochemical staining showed that TIMP-1 related antigen in livers was located in cytoplasm. The proteinexpression of TIMP-1 was significantly higher in the oldestanimals and the mRNA expression was increased significantlyin the 24-mo-old rats (t= 4.61, P= 0.002＜0.05, 24-vs 10-mo-old rats; t= 4.31, P= 0.003＜0.05, 24- vs 3-mo-oldrats). The expression of MMP-2 and MMP-9 had no change during aging; the ratios TIMP-1/MMP-2 and TIMP-1/MMP-9 in aging liver were significantly higher than those in maturation and young livers.CONCLUSION: TIMP-1 may play an important role in the process of liver aging.

Glycyrrhizinate reduces portal hypertension in isolated perfused rat livers with chronic hepatitis

AIM:To investigate the effects of diammonium glycyrrhizinate(Gly)on portal hypertension(PHT)in isolated portal perfused rat liver(IPPRL)with carbon tetrachloride(CCl4)-induced chronic hepatitis.METHODS:PHT model was replicated with CCl4 in rats for 84 d.Model was identified by measuring the ascetic amounts,hepatic function,portal pressure in vivo,splenic index,and pathological alterations.Inducible nitric oxide synthase(iNOS)in liver was assessed by immunohistochemistry.IPPRLs were performed at d0,d28,d56,and d84.After phenylephrine-induced constriction,Gly was geometrically used to reduce PHT.Gly action was expressed as median effective concentration(EC50)and area under the curve(AUC).Underlying mechanism was exploited by linear correlation between AUC values of Gly and existed iNOS in portal triads.RESULTS:PHT model was confirmed with ascites,splenomegaly,serum biomarkers of hepatic injury,and elevated portal pressure.Pathological findings had shown normal hepatic structure at d0,degenerations at d28,fibrosis at d56,cirrhosis at d84in PHT rats.Pseudo lobule ratios decreased and collagen ratios increased progressively along with PHT development.Gly does dose-dependently reduce PHT in IPPRLs with CCl4-induced chronic hepatitis.Gly potencies were increased gradually along with PHT development,characterized with its EC50at 2.80×10-10,3.03×10-11,3.77×10-11and 4.65×10-11mol/L at d0,d28,d56and d84,respectively.Existed iNOS was located at hepatocyte at d0,stellate cells at d28,stellate cells and macrophages at d56,and macrophages in portal triads at d84.Macrophages infiltrated more into portal triads and expressed more iNOS along with PHT development.AUC values of Gly were positively correlated with existed iNOS levels in portal triads.CONCLUSION:Gly reduces indirectly PHT in IPPRL with CCl4-induced chronic hepatitis.The underlying mechanisms may relate to rescue NO bioavailability from macrophage-derived peroxynitrite in portal triads.

Changes of Serum Trace Elements, AFP, CEA, SF, T3, T4 and IGF-Ⅱ in Different Periods of Rat Liver Cancer

Objective: Based on liver cancer model built in SD rats, the contents of trace elements (Cu, Fe, Zn, Ca and Mg), AFP, CEA, SF, TH and IGF-II in serum were measured at different stages to explore the molecular changes during the rat liver cancer development. Methods: The SD rat liver cancer model was built by using diethylnitrosamine (DENA) as the mutagen. During 16 weeks of DENA gavage, blood samples were taken in the 14th, 28th, 56th, 77th, 105th and 112th days respectively after the first day of gavage with DENA, then the contents of five trace elements (Cu, Fe, Zn, Ca and Mg), T3, T4, IGF-II, AFP, CEA and SF in serum were determined. Results: During the development of the rat liver cancer, in the test group, the Cu content significantly increased in serum, while the contents of Fe, Zn and Ca significantly decreased. The content of Mg showed no significant change. AFP and CEA of the test group showed same expression level with the control group; while the content of SF was lower than that of the control group when cancerization appeared. T3 and T4 increased at the first stage and then went down, and the content of IGF-II was always high. Conclusion: Cu, Fe, Zn, Ca, T3, T4, SF and IGF-II are closely related to the development of liver cancer. The changes of their contents in the development of cancer could enlighten the researches on cancer pathogenesis and prevention.

Protective effect of zinc: a potent heat shock protein inducer in cold preservation of rat liver

Objective: To study the synthesis of heat shock pro- tein 70 (HSP70) by zinc ( ZnSO_4) and its protective effect during cold preservation in rat liver by estab- lishment of a simple cold preservation model. Methods: Wistar rats were divided into 5 groups (n =6): control group untreated before operation; Zn- 1, Zn-2, and Zn-3 groups treated before operation with zinc sulfate (Zn^(2+) 5 mg/kg, 10 mg/kg, 15 mg/ kg respectively); H group treated with heat shock (42.5 ℃×15 min). The livers of the rats were pre- served in UW solution for 6, 12, 24 hours. The re- sults of heat shock protein (HSP) synthesis were ana- lyzed with Western blot. The aspartate aminotrans- ferase (AST) and lactic dehydrogenase (LDH) values of perfusion and histological findings were evaluated. Results: A great amount of HSPs was synthesized af- ter pretreatment with zinc and heat shock. The AST and LDH values of the Zn-1, Zn-2 and H groups were significantly lower than those of the C group (P <0.05). But the value of the Zn-3 group was much higher. Histologically, mild injury was observed in the Zn-1, Zn-2 and H groups, but severe injury in the Zn-3 group. Conclusions: Zn^(2+), as a potent and feasible inducer of HSP expression, is able to protect the liver for cold preservation. Proper induction dosage of Zn^(2+) ranges from 5 mg/kg to 10 mg/kg, and Zn^(2+) 15 mg/ kg could not be a stress conditioning factor for its ad- verse effect on rat liver.

The effect of ischemic precondition to IL-6 on rat liver ischemiareperfusion injury in transplantation

Objective:To investigate the effect of ischemic precondition to protect ischemia-reperfusion injury and reduce IL-6 expression in the rats liver transplantation.Methods:The rat portal vein infusion of autologous liver transplantation model were used.The rats were divided into ischemic preconditioning rats liver transplantation group(A group),the rats liver transplantation group(B group) and the normal rat control group(C group).Then we analyzed the changes of liver function,liver microstructure and the expression of IL-6,SOD and MDA within 48 h.Results: The pathology of liver in group A showed lobular architecture essentially normal,the liver cells was slightly swell and no significant changes in postoperative 12 h.In transmission electron microscope(46 000X).the mitochondria of liver cells in group A i】ecame swelling,elliptical can cristae partially broken.But there still has a small amount of arrangement.While that in group, the mitochondria were swollen,became round,serious visible crest reduce or ruptured.The result of over function test showed that the serum ALT and AST levels in group A and B were both higher than that in group C at each time period,but the serum ALT and AST levels in group A were lower than that in group B.The expression changes of IL-6 in group B were higher than that in group A and R(P【0.05).The expression of MDA in group A is more obvious than that in group B(P【0.05).Conclusions:Ischemic precondition could alleviate part of ischemia-reperfusion injury in the rat liver transplantation,and also could reduce IL-6 expression to protect the liver cells against liver damage and inflammatory cytokine production.

Failure of P-selectin blockade alone to protect the liver from ischemia-reperfusion injury in the isolated blood-perfused rat liver

AIM:To determine if blockade of P-selectin in the isolated blood-perfused cold ex vivo rat liver model protects the liver from ischemia-reperfusion injury. METHODS:The effect of P-selectin blockade was assessed by employing an isolated blood-perfused cold ex vivo rat liver with or without P-selectin antibody treatment before and after 6 h of cold storage in University of Wisconsin solution. RESULTS:In our isolated blood-perfused rat liver model,pre-treatment with P-selectin antibody failed to protect the liver from ischemia-reperfusion injury,as judged by the elevated aspartate aminotransferase activity. In addition,P-selectin antibody treatment did not significantly reduced hepatic polymorphonuclear leukocyte accumulation after 120 min of perfusion. Histological evaluation of liver sections obtained at 120 min of perfusion showed significant oncotic necrosis in liver sections of both ischemic control and P-selectin antibody-treated groups. However,total bile production after 120 min of perfusion was signifi cantly greater in P-selectin antibody-treated livers,compared to control livers. No signifi cant difference in P-selectin and ICAM-1 mRNAs and proteins,GSH,GSSG,and nuclear NF-κB was found between control and P-selectin antibody-treated livers. CONCLUSION:In conclusion,we have shown that blockade of P-selectin alone failed to reduced polymorphonuclear leukocyte accumulation in the liver and protect hepatocytes from ischemia-reperfusion injury in the isolated blood-perfused cold-ex vivo rat liver model.

Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models

瞄准：在老鼠在导致免疫者、导致 CCL4 的肝纤维变性模型评估在 TIMP-1 表示和肝纤维变性之间的浆液 TIMP-1 水平和关联。方法：导致免疫者、导致 CCL4 的肝纤维变性模型被地塞米松(0.01 mg ) 和 CCL4 分别地建立。浆液 TIMP-1 水平与 ELISA 被检测，当肝活体检视的组织病理学说的等级被评估时。枪兵等级关联测试被用来在二个纤维变性模型分析在 TIMP-1 表示和肝的纤维变性之间的关联的差别。而且，原位杂交被用来在二个模型决定 TIMP-1 mRNA 的表示差别。结果：积极关联在有免疫力的劝诱的组的浆液 TIMP-1 水平和相应老鼠的纤维变性肝的组织病理学说的阶段之间存在(枪兵等级关联测试， r = 0.812， P 【 0.05 ) ，并且 TIMP-1 mRNA 的积极原位杂交信号是强壮的。在导致 CCL4 的肝纤维变性模型，在浆液 TIMP-1 水平和肝的纤维变性的严厉之间的关联不是统计上重要的(枪兵等级关联测试， r = 0.229， P 】 0.05 ) 。并且与导致免疫者的模型，相比， TIMP-1 mRNA 的积极原位杂交信号是更弱的，当表达式变化在一样的严厉的肝的纤维变性是更高的时。结论：在在二个老鼠肝纤维变性模型的 TIMP-1 表示和肝纤维变性之间的关联是不同的。在导致免疫者的模型，，浆液 TIMP-1 水平能在导致 CCL4 的模型，反映肝纤维变性的严厉在浆液 TIMP-1 水平和肝的纤维变性的严厉之间的关联不是统计上重要的。

The study on effect of long-termed administration of mixed rare earth Changle on rat liver

目的 :研究不同剂量的“常乐”对大鼠肝脏结构和功能的影响。方法 :采用健康 Wistar大鼠 1 80只 ,每组 30只 ,实验组分别以 0 .1、0 .2、2 .0、1 0 .0、2 0 .0 mg· kg-1常乐连续灌胃 6个月 ,每日 1次。应用常规组织化学、透射电镜技术及血清生化测定技术 ,观察动物肝脏的结构和功能变化。结果 :各实验组动物体重随染毒剂量的减少而呈线性增长。 2 0 .0 mg·kg-1组肝细胞内糖原颗粒减少 ,肝细胞核有不同程度的变形 ,门管区有炎细胞浸润 ,其余各组肝脏结构与对照组相比无明显改变。各实验组动物肝脏 Kupffer细胞及肝细胞中均可见到高电子密度的致密体及含电子密度较高颗粒的溶酶体 ,其数量随染毒剂量的增大而增多。 2 0 .0 mg· kg-1组血清 ALP及 GPT增高。结论 :2 0 .0 mg·kg-1“常乐”长期作用对大鼠肝脏结构和功能均有损害。

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver

AIM To develop a simplified bioartificial liver(BAL) device prototype, suitable to use freshly and preserved liver Microorgans(LMOs) as biological component. METHODS The system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hema-tocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and Via Span~? solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase(LDH) release and ammonia detoxification capacity. RESULTS The concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and Via Span~? were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls(49.3% ± 8.8%, P < 0.05). LDH release was 6.0% ± 2.3% for control LMOs, and 6.2% ± 1.7% and 14.3% ± 1.1% for BG35 and Via Span~? cold preserved LMOs, respectively(n = 6, P < 0.05). CONCLUSION This prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

Protective effects of N-acetylcysteine on brain-dead rat liver

BACKGROUND: Brain-dead donors have been the main sources in organ transplantation. But many studies show that brain-death affects the organ's function after transplantation. This study was undertaken to investigate liver injury after brain-death in rats and the protective effects of N-acetyleysteine (NAC) on liver injury. METHODS: A total of 30 Wistar rats were randomized into 3 groups: normal control group (C), brain-dead group (B), and NAC pretreatment group (N). At 4 hours after the establishment of a brain-dead model, serum was collected to determine the levels of ALT, AST, TNF-αand hyaluronic acid (HA). Hepatic tissue was obtained for electron microscopic examination. RESULTS: At 4 hours, the levels of ALT, AST, TNF-a, and HA in group N were significantly higher than those in group C, but these parameters were significantly lower than those in group B. Electron microscopy showed activated Kupffer cells, denuded sinusoidal endothelial cells (SECs), and widened fenestration in group B, but eliminated activation of Kupffer cells and intact SECs in group N. CONCLUSION: Brain death can cause liver injury, and N-acetyleysteine can protect the liver from the injury.