HISTOLOGICAL AND BIOCHEMICAL EVALUATION OF PRECISION-CUT LIVER SLICES

Human and animal precision-cut tissue slices are being widely used to obtain drug metabolism and toxicity profiles in vitro. These data are then used to predict what might be seen in human patients. The accuracy of the extrapolation of findings based on human or animal in vitro systems to the findings that occur in vivo is dependent on the quality of the in vitro system. This study investigates the optimal thickness of tissue slices and the optimal incubation system, as determined by histological evaluation (light and electron microscopy) and two biochemical parameters (adenosine triphosphate [ATP] content and potassium [K +] retention). The three incubation systems tested were the dynamic organ culture system and the roller incubation system for volatiles, which are surface culture systems, and the multiwell plate culture system, which is a submersion culture system. The cellular glycogen content of canine liver slices was determined using slices fixed in 10% buffered formalin and a periodic-acid Schiff stain. After determining the optimal slice thickness (≈150 μm) and incubation system (the dynamic organ culture system) using gross histological evaluation, the liver slices were incubated for 6, 12, 24, 48, and 72 h, and the slices were evaluated using electron microscopy. The micrographs showed that the hepatocytes displayed normal morphology for up to 72 h. These histological findings were compared to the biochemical findings. ATP content was found to be more sensitive in detecting cellular degeneration than K + retention but less sensitive than the histological evaluation. The use of tissue slices that are produced and incubated under optimal conditions can be a reliable in vitro method of investigating morphologic and metabolic alterations produced by a variety of agents, including drugs and toxicants.     

TOXICITY OF A SEVOFLURANE DEGRADATION PRODUCT INCUBATED WITH RAT LIVER AND RENAL CORTICAL SLICES

Compound A (2-fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene) is a degradation product of the anesthetic sevoflurane which is created in closed-circuit anesthetic machines. Past in vivo and in vitro studies have implied that Compound A is nephrotoxic via bioactivation through the cysteine conjugate β-lyase pathway. Although glutathione (GSH) conjugates of Compound A have been reported, it is not clear if they are formed enzymatically or via direct reaction with GSH. To determine if these metabolites are produced and toxic, a tissue slice system that first exposes male Fischer 344 rat liver slices to volatilized Compound A followed by exposure of rat kidney slices to the liver incubate was employed. Liver slices exposed to volatilized Compound A (6–12 μM medium conc.; ～23 ppm) exhibited a loss of K⁺ by 6 h, which was not seen in kidney slices exposed to Compound A. Aminobenzotriazole, a cytochrome P 450 suicide inhibitor, initially inhibits the cytotoxicity of Compound A to liver slices (at these times and concentrations). The sequential liver/kidney slice experiments using Compound A have not demonstrated nephrotoxic results. GSH conjugates were synthesized and was found to be nephrotoxic at concentrations above 91 μM (18 h), with higher concentrations showing toxicity at earlier times. Additionally, non-enzymatic reactions of Compound A with GSH or sulfhydryl-containing medium produces nephrotoxic products. These studies show that Compound A is directly toxic to the liver, possibly via P 450 activation, and Compound A can react with sulfhydryls directly to produce a nephrotoxic.     

DRINKING PATTERNS AND BEVERAGE PREFERENCES OF LIVER CIRRHOSIS PATIENTS IN MEXICO

The purpose of this study was to investigate the pattern of alcoholism in a special group of alcoholics (alcoholic cirrhotics) in a hospital-based population in west central México and assess the role of regional spirits such as tequila. A complete alcohol drinking history and a structured questionnaire directed at investigating the pattern of alcohol consumption was applied to124 adult patients with chronic liver disease caused by alcohol during January1995 to January 1996.

The mean age of onset was 27 ± 3 years in women and 18 ± 0.5 years in men. The mean alcohol intake per week was 749 ± 192g for women and 1113 ± 151g for men. On average, patients consumed alcohol for a mean of 24.5 years. The overall patient drinking preference was for tequila followed by 96° Gay Lusac (G.L.), alcohol, and beer. In a subset of 70 patients three phases of alcoholism could be identified (prealcoholic, critical, and chronic). Each phase had a mean duration of at least 11 years.Beer was the dominant beverage in the prealcoholic phase while tequila was consumed more often in the other phases. In the critical phase of alcoholism an average of 337g of alcohol were consumed per week and in the chronic phase1765g/week.

Tequila was the overall preferred beverage in this group of alcoholics.Other beverages included beer and straight alcohol with a clear trend from less to higher concentration of alcohol throughout the drinking history. Subtle gender differences in the patterns of alcoholism may be suspected. In this group of patients the role of tequila drinking is highlighted.     

硒和大蒜素对糖尿病小鼠肝脏生化功能改变的保护作用

目的：观察亚硒酸钠、大蒜素及其合并用药对糖尿病小鼠肝脏生化功能改变的保护作用。方法：建立四氧嘧啶糖尿病小鼠动物模型，测定肝脏抗氧化酶、ATP酶活性、脂质过氧化产物、一氧化氮（NO）和肝糖原含量。结果：糖尿病小鼠肝脏抗氧化功能和Na^2 ,K^ -ATPase的活性明显下降，NO和肝糖原含量明显减少，与糖尿病模型组小鼠相比，硒、大蒜素能明显逆转上述变化，其中硒对GSHpx,Na^ ,K^ -ATPase,Ca^2 -ATPase活性的作用强于大蒜素，大蒜素对SOD活性的影响较大；硒、大蒜素合并用药较单独用药作用减弱。结论：硒、大蒜素对糖尿病小鼠肝脏具有良好的保护作用，两药合用作用减弱。     

水飞蓟宾对小鼠肝脏炎症损伤和肿瘤坏死因子的产生及活性的影响

建立了小鼠肝损伤模型及体内外肿瘤坏死因子(TNF)诱生的方法,研究水飞蓟宾(SB)对肝脏炎症损伤、TNF产生及其生物活性的影响。结果表明∶SB在体内外均可显著抑制脂多糖(LPS)诱导TNF产生;在体外能抑制TNF对肝细胞系GSG-7701和成纤维细胞系L929的细胞毒作用;在体内对LPS诱导的痤疮丙酸杆菌致敏的小鼠肝脏炎症损伤有保护作用。提示SB的保肝作用机理与其抑制TNF的产生和活性有关。     

原卟啉对家兔、小鼠肝脏损害的保护作用

原卟啉对CCl_4所致的家兔急性肝损伤,经组织学检查具有明显的保护作用,可使家兔SGPT、SGOT、γ—GT显著下降,或趋于正常,以及调节血中氨基酸代谢;并可增加小鼠肝脏血流量和促进肝细胞DNA蛋白质的合成。     

齐墩果酸防治实验性肝损伤作用的研究

齐墩果酸对CCl₄引起的大鼠急性肝损伤有明显的保护作用。表现在药物能使血清GPT明显下降;肝内甘油三酯蓄积量减少;肝细胞变性、坏死明显减轻,糖原蓄积增加;根据超微结构观察,肝细胞内线粒体肿胀与内质网囊泡变减轻。     

紫堇灵、乙酰紫堇灵及原鸦片碱对小鼠实验性肝损伤的保护作用

研究了紫堇灵、乙酰紫堇灵及原鸦片碱对小鼠实验性肝损伤的保护作用及其作用机理。小鼠预先分别ｉｇ紫堇灵、乙酰紫堇灵或原鸦片碱５０及１００ｍｇ·ｋｇ－１２次，对ＣＣｌ４、硫代乙酰胺、扑热息痛所致的小鼠肝损伤均有保护作用，使ＳＧＰＴ显著降低，肝病理损伤程度减轻。此３种成分在体外均能抑制ＣＣｌ４引起的肝微粒体脂质过氧化及ＣＣｌ４转化为ＣＯ。在上述实验中乙酰紫堇灵的作用均强于另外两种成分。另外，此３种成分对肝药酶有先抑制后诱导作用。     

紫堇灵、乙酰紫堇灵及原鸦片碱对小鼠实验性肝损伤的保护作用

研究了紫堇灵、乙酰紫堇灵及原鸦片碱对小鼠实验性肝损伤的保护作用及其作用机理。小鼠预先分别ig紫堇灵、乙酰紫堇灵或原鸦片碱50及100mg·kg^-12次,对CCl₄、硫代乙酰胺、扑热息痛所致的小鼠肝损伤均有保护作用,使SGPT显著降低,肝病理损伤程度减轻。此3种成分在体外均能抑制CCl₄引起的肝微粒体脂质过氧化及CCl₄转化为CO。在上述实验中乙酰紫堇灵的作用均强于另外两种成分。另外,此3种成分对肝药酶有先抑制后诱导作用。     

肝匀浆对双呋啶的体外活化作用

本实验提示新鲜人肝匀浆在体外可将双呋啶活化为具有抗癌活性的氟脲嘧啶。活化产物经紫外光谱测定,于265nm 处出现一吸收峰,与氟脲嘧啶的特征吸收峰吻合。温育60分钟,氟脲嘧啶含量达0.0061g/L。未加肝匀浆对照管氟脲嘧啶含量几乎测不出。溴液褪色试验阳性。金黄色葡萄球菌抑菌试验阳性。本实验证明猪肝匀浆与人肝匀浆同样具有潘化双呋啶的作用,并为体内活化的抗癌药的体外药敏试验提供了前提条件。     

CARDIOVASCULAR AND RESPIRATORY EFFECTS OF CAROTID BODY STIMULATION IN THE MONKEY

1. The carotid bodies were stimulated in the anaesthetized pig-tailed macaque monkey (M. nemestrina) using (i) brief injections of cyanide or CO₂-equilibrated bicarbonate solution into a common carotid artery, and (ii) longer perfusion with hypoxic hypercapnic blood in vascularly isolated chemoreceptor preparations. 2. In spontaneously breathing animals brief stimuli (thirty-one tests, seven monkeys) consistently increased pulmonary ventilation (by 97 ± 10% of control), slowed the heart rate (the pulse interval increasing by 36 ± 7.5%), and increased femoral vascular resistance (by 44 ± 7%). 3. More sustained chemoreceptor stimulation with asphyxial blood (nineteen tests, five monkeys) increased ventilation by 187 ± 23%, but transient bradycardia occurred in only eight of nineteen tests and was followed by tachycardia; in the remaining tests, only tachycardia occurred. After 20–40 s, the pulse interval was 5.8 ± 0.9% below the control level. Femoral vascular resistance either increased (five tests, two animals) or decreased (six tests, two animals). 4. Evidence is presented that in the monkey the autonomic effects of chemoreceptor stimulation are influenced by the level of respiratory activity with bradycardia and vasoconstriction occurring when the level is low, and tachycardia and vasodilatation when it is high. 5. The interaction of autonomic responses resulting from carotid body stimulation and from mechanisms initiated by the concomitant hyperventilation are qualitatively similar in the monkey and in subprimate species, although there may be quantitative differences such as would account for the species differences to disturbances produced, for instance, by arterial hypoxia.     

姥鲨鱼肝油乳抗肿瘤作用的研究

使用30%姥鲨肝油乳剂在15-30ml/kg的剂量范围内,通过对S_(180)、HePA、Lewis等三种小鼠移植性实体型肿瘤实验,最高抑制率分别为58%、55%、49%。     

米托蒽醌聚乳酸缓释毫微粒冻干针剂在动物体内的靶向性研究

目的：比较肝靶向米托蒽醌聚乳酸缓释毫微粒（ＤＨＡＱ－ＰＬＡ－ＮＰ）冻干针剂和ＤＨＡＱ水针剂在小鼠体内的分布规律,验证前者的肝靶向性。方法：采用ＨＰＬＣ法测定静注ＤＨＡＱ－ＰＬＡ－ＮＰ和ＤＨＡＱ水针剂后小鼠血液、心、肝、脾、肺、肾的药物浓度,由此计算各器官的相对百分含量。结果：ＤＨＡＱ－ＰＬＡ－ＮＰ冻干针剂在肝脏的分布明显高于ＤＨＡＱ水针剂,在其它器官中的含量则低于水针剂,给药２４小时后药物在肝中的     

人参茎叶皂甙(SSLG)对实验性肝损伤的影响

SSLG可抑制CCl₄和硫代乙酰胺中毒小鼠SGPT的升高和肝中P-450、RNA及糖元含量的降低。此外,SSLG还能降低中毒大鼠肝中脂肪和羟脯氨酸的含量。对于小鼠的免疫性肝损伤,SSLG仅能使血中LDH-5含量降低,而对血清免疫复合物含量及组织学改变均无影响。SSLG还可诱导正常或中毒小鼠肝脏P-450含量增加及缩短小鼠的戊巴比妥钠睡眠时间,并能促进肝脏排泄BSP的功能。上述结果表明,SSLG对实验性肝损伤具有一定的保护作用。     

CHARACTERIZATION AND REGULATION OF RAT LIVER UDP GLUCURONOSYLTRANSFERASES

1. The use of DNA recombinant techniques to study UDP glucuronosyltransferases is reviewed. 2. The cloning and sequencing of the cDNAs to five forms of UDP glucuronosyltransferase demonstrated that these proteins are encoded by a superfamily of genes. 3. The substrate specificities of four isozymes have been analysed by expression of their corresponding cDNAs in cell culture. 4. Analysis of liver mRNA levels using radiolabelled DNA probes demonstrated that one isozyme, UDPGTr-2, is elevated by phenobarbital whereas a second isozyme, 4-nitrophenol GT is elevated by 3-methylcholanthrene. The levels of other isozymes are unaffected by these two prototypic inducers of drug metabolizing enzymes. 5. The level of 4-nitrophenol GT mRNA is elevated 10-15-fold in hyperplastic nodules in livers of rats treated with 2-acetylaminofluorene. Similar increases were not observed for mRNAs encoding other forms of UDP glucuronosyltransferase.     

肝靶向羟基喜树碱缓释毫微粒的研究

采用吸附—包裹法制备了聚乙烯吡啶烷酮包被的羟基喜树碱聚氰基丙烯酸正丁酯毫微粒。研究了该毫微粒的形态、粒径及粒径分布、载药量、体外释药特征、动物体内的分布与药代动力学参数。结果表明平均粒径dav=81.2nm,载药量为1.22%,体外释药速率符合Higuchi方程:Q=0.0615+0.0940t,静脉注射后15min,即有68.2%羟基喜树碱浓集于肝脏。血浆药浓—时间曲线符合二室开放药动学模型。主要药动学参数为:Vc=3.548L,T_1/2β=146.99h,CL=0.1788L·h^-1。说明该载药毫微粒具有明显的肝靶向和缓释作用。本文报道的吸附─包裹法对水、脂不溶性药物聚氰基丙烯酸酯毫微粒的制备具有一定参考价值。     

我国成人肝微粒体的制备和几种细胞色素P450单加氧酶的活性

7例人肝标本均取自我国成年男性,其中5例来自非疾病死亡者,1例为肝血管瘤手术切除的正常肝组织,1例取自交通事故死亡者。该7例人肝微粒体细胞色素P450和细胞色素b5含量分别为0.36±0.08和0.23±0.05nmol·mg~(-1)蛋白,氨基比林和乙基吗啡N-脱甲基酶活性分别为1.07±0.23和1.82±0.31nmol·mg~(-1)·min~(-1),7-乙氧基香豆素O—脱乙基酶、硝苯吡啶氧化酶和(-)-吡喹酮羟化酶活性分别为0.30±0.10,0.43±0.18和0.69±0.43nmol.mg~(-1)·min~(-1)。     

博来霉素A6抗人体肝癌的实验研究

用克隆形成测定法检查博来霉素A₆对人肝癌、鼻咽癌和胃癌等细胞系的杀伤作用,发现对肝癌BEL-7402细胞有高度活性,半数杀伤浓度(IC₅₀)为6×10^-11mol/L。用流式细胞光度术研究表明,博来霉素A₆对肝癌BEL-7402细胞有G₂期阻断作用。在裸鼠可以耐受的剂量下,博来霉素A₆对移植性人肝癌的肿瘤生长有显著抑制作用,抑制率为75%。研究结果提示博来霉素A₆有可能用于肝癌化疗。     

PRIMARY STRUCTURE OF THE ACTIVE TRYPTIC FRAGMENTS OF HUMAN AND MONKEY SALIVARY ANIONIC PROLINE-RICH PROTEINS

The primary structures of the four human salivary anionic proline-rich proteins and an analogous protein from the saliva of a monkey (Macaca arctoides) have been further investigated. These proteins possess the unusual property of inhibiting crystal growth of calcium phosphate salts, and it has been proposed that they play an important role in the mouth, by preventing precipitation of calcium phosphate salts from the supersaturated saliva. The tryptic fragments responsible for this activity have been isolated from all five proteins and their complete amino acid sequences determined and compared. These active fragments have been unequivocally identified as the amino-terminal segment in all five proteins. The structures of the four human fragments are identical except for the presence of Asn at residue 4 in PRP-1 and -3 instead of Asp found in PRP-2 and -4. Comparison of the 30 residue human fragments with the monkey fragment shows 18 residues to be identical in these peptides, providing that residue 1 of the monkey fragment is aligned with residue 3 of the human proteins. These studies constitute the next step in determining the mechanism of action of these unusual proteins, and in determining their minimum chain length required for inhibitory activity.     

我国成人肝微粒体的制备和几种细胞色素P450单加氧酶的活性

7例人肝标本均取自我国成年男性,其中5例来自非疾病死亡者,1例为肝血管瘤手术切除的正常肝组织,1例取自交通事故死亡者。该7例人肝微粒体细胞色素P450和细胞色素b5含量分别为0.36±0.08和0.23±0.05nmol·mg^-1蛋白,氨基比林和乙基吗啡N-脱甲基酶活性分别为1.07±0.23和1.82±0.31nmol·mg^-1·min^-1,7-乙氧基香豆素O—脱乙基酶、硝苯吡啶氧化酶和(-)-吡喹酮羟化酶活性分别为0.30±0.10,0.43±0.18和0.69±0.43nmol.mg^-1·min^-1。