Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration

The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied. After a single oral dose of dimethylnitrosamine (2–75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease. Nucleosomes were separated on sucrose density gradients. There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals. The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 g _av) and nucleosomal peak fractions were used for isolation of DNA. DNA was heat denatured in 7 M urea or formamide. After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area. The increase in DNA fragmentation was dimethylnitrosamine dose response dependent. When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight. A good correlation between mobility and log of chain length of 174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea. DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides. Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin. It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.Abbreviations DMN dimethylnitrosamine - SDS sodium dodecyl sulfate The work was supported by Grant Number 1 R0 1 CA26642-01, awarded to A.v.d.D. by the National Cancer Institute, DHEW     

The anti-fibrogenic effect of a pharmaceutical composition of [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] (oltipraz) and dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB)

Liver fibrosis is a prepathological state wherein damaged liver tissues in chronic liver diseases, such as hepatitis, are not repaired to normal tissues, but converted to fibrous tissue. 5-(2-Pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz), a cancer chemopreventive agent, is effective against a wide variety of chemical carcinogens. Recently, we reported that oltipraz inhibits liver fibrogenesis (Kang et al., 2002). In the present study, the effects of oltipraz in combination with dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) on dimethylnitrosamine (DMN)-induced liver fibrogenesis were assessed in rats. Oltipraz (30 mg/kg body weight, p.o., 3 times per week for 4 weeks) was found to inhibit the increases in plasma ALT, AST and bilirubin by DMN, whereas DDB (30 mg/kg body weight, p.o., 3 times per week for 4 weeks) attenuated the increases in the plasma ALT and bilirubin. The lowered plasma protein and albumin contents in DMN-treated rats were completely restored by oltipraz, but not by DDB. DDB decreases liver cell injury and inflammation through inhibition of nuclear factor-kB. DMN increased the accumulation of liver collagen, as indicated by the increase in the 4-hydroxyproline content in liver homogenates, which was reduced by treatment with oltipraz, but not by DDB. Given the differential effect between oltipraz and DDB, the potential enhancement of antifibrotic efficacy by the drugs was assessed in the animal model. Despite the minimal effect of DDB on DMN-induced fibrogenesis, DDB (5-25 mg/kg), administered together with oltipraz (25-5 mg/kg), showed an additive protective effect against hepatotoxicity and fibrosis induced by DMN, which was shown by the blood chemistry parameters and histopathological analysis. The adequate composition ratio of oltipraz to DDB was 5:1. These results provide information on the pharmaceutical composition, comprising of oltipraz and DDB as the active components, for the treatment and/or prevention of liver fibrosis and cirrhosis.     

阿米洛利联用苦参素预防大鼠肝纤维化的实验

目的观察阿米洛利联用苦参素预防大鼠肝纤维化的效果。方法40只大鼠随机分为正常对照组（n＝5）、模型对照组(n＝7)、阿米洛利组（n＝7）、苦参素组（n＝7）、联合药物组（n＝7）和阳性对照组（n＝7）。除正常对照组外，其他各组每周开始3 d连续腹腔注射二甲基亚硝胺10 mg·kg 1·d-1，共3周，建立大鼠肝纤维化模型。阿米洛利组造模同时每日灌胃阿米洛利1 mg·kg 1，苦参素组每日灌胃苦参素500 mg·kg 1，联合药物组每日灌胃阿米洛利和苦参素，剂量与单用药组相同，共用药3周；阳性对照组造模同时每日灌胃马洛替酯90 mg·kg 1。正常对照组每周连续3 d腹腔注射0.9%氯化钠溶液，共3周，同时每日灌胃等量0.9%氯化钠溶液共3周。观察各组大鼠肝重/体重和脾重/体重比值，血及肝组织生化，羟脯氨酸（HYP）、玻璃酸（HA）及层黏蛋白（LN）含量的变化。结果苦参素组和联合药物组及阳性对照组较模型对照组肝功能得到改善（P＜0.01）；阿米洛利组和苦参素组较模型对照组血清HA、LN和HYP含量降低（P＜0.05）；4个给药组肝组织内超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH PX）较模型对照组升高，而丙二醛（MDA）低于模型组（P＜0.01）；联合药物组效果最佳。结论阿米洛利与苦参素对二甲基亚硝胺诱导的肝纤维化均有预防作用，两者联用有一定的协同作用。     

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats

Summary Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any wellknown organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.Abbrevations DMN dimethylnitrosamine - F-344 Fischer-344 - WGA wheat germ agglutinin - PNA Arachis hypogaea (peanut) agglutinin - LTA Lotus tetragonolobus agglutinin     

Iron deposition and fat accumulation in dimethylnitrosamine-induced liver fibrosis in rat

AIM： To investigate if iron deposition and fat accumulation in the liver play a pathogenetic role in dimethylnitrosamine （DMN）-induced liver fibrosis in rat.
METHODS： Thirty rats were treated with DMN at does consecutive days of 10 μL/kg daily, i.p., for 3 consecutive day each week for 4 wk. Rats （n = 30） were sacrificed on the first day （model group A） and 21st d （model group B） after cessation of DMN injection. The control group （n = 10） received an equivalent amount of saline. Liver tissues were stained with hematoxylin ＆ eosin （HE） and Masson and Prussian blue assay and oberserved under electron microscopy. Serum alanine aminotransferase （ALT）and liver tissue hydroxyproline （Hyp） content were tested.
RESULTS： The liver fibrosis did not automaticallyreverse, which was similar to previous reports, the perilobular deposition of iron accompanied with collagen showed marked characteristics at both the first and 21st d after cessation of DMN injection. However, fat accumulation in hepatocytes occurred only at the 21^st d after cessation of DMN injection.
CONCLUSION： Iron deposition and fat accumulation may play important roles in pathological changes in DMN-induced rat liver fibrosis. The detailed mechanisms of these characteristics need further research.     

Hepatoprotective effects of Yi Guan Jian, an herbal medicine, in rats with dimethylnitrosamine-induced liver fibrosis

Aims of the study

Yi Guan Jian (YGJ) has long been employed clinically to treat liver fibrosis in traditional Chinese Medicine but the mechanism underlying the regulation has not been clarified in detail. The present investigation was designed to assess the involvement of the fibrosis pathway in dimethylnitrosamine (DMN)-induced liver fibrosis in rats.

Materials and methods

Liver fibrosis was induced by DMN injection (10 mg/kg, i.p., given three consecutive days each week) following 4 weeks. YGJ was oral administered (1.8 g/kg daily via gastrogavage for two weeks). Liver sample were subjected to histological and western blot studies. For evaluation of hepatic fibrosis-related factors, collagen α1-I, tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-smooth muscle actin (α-SMA) mRNA and protein levels were analyzed.

Results

YGJ remarkably prevented body weight loss and DMN damage in the liver, and it inhibited the elevation of serum glutamate oxaloacetate transaminase (GOT), and glutamic pyruvic transaminase (GPT). Oral administration of YGJ extract significantly reduced the accumulation of collagen α1-I, TIMP-1, and α-SMA in liver tissues.

Conclusions

Taken together, these findings indicate that the YGJ Chinese herb showed hepatoprotective and anti-fibrogenic effects against DMN-induced hepatic injury. Our data suggest that the YGJ may be useful in reversing the development of hepatic fibrosis.     

Effect of chronic alcohol consumption on tumor incidence due to dimethylnitrosamine administration

Summary To study the effect of chronic alcohol consumption on tumor development due to dimethylnitrosamine (DMN) administration, female Sprague-Dawley rats were pair-fed for 3 weeks a nutritionally adequate liquid diet containing either ethanol (36% of total calories) or isocalorically substituted carbohydrates as control diet. Thereafter, the animals were maintained on laboratory chow and tap water ad libitum for another 2 weeks and received 1.5 mg DMN i.p. per day for the first 5 days. This 5-week cycle was repeated three more times. Chronic treatment with an alcohol-containing diet was shown to significantly improve the mean survival time of DMN-treated rats compared with identically treated animals fed the control diet, but the total number of tumors observed under these experimental conditions and the target organ remained virtually unchanged.Abbreviations DMN dimethylnitrosamine - DEN diethylnitrosamine     

胰腺弹力蛋白酶在大鼠肝纤维化组织中的表达

目的:观察胰腺丝氨酸弹力蛋白酶的基因、蛋白及活性在大鼠肝纤维化中的表达,探讨弹力蛋白酶在肝纤维化形成中的作用.方法:采用二甲基亚硝胺(DMN)4周12次腹腔注射制备Wistar雄性大鼠肝纤维化模型,以正常组作对照.观察肝功能,肝脏病理组织学,肝组织羟脯氨酸含量以及肝组织弹力蛋白酶mRNA、蛋白及其活性的表达.结果:弹力蛋白酶mRNA、蛋白及其活性在正常大鼠肝组织均有一定程度的表达,在肝纤维化模型组,假小叶内肝细胞弹力蛋白酶mRNA表达有所减少,弹力蛋白酶活性在正常组和模型组分别为(11.43±2.35)U/g、(8.02±1.62)U/g,模型组显著降低(P＜0.05).结论:某种程度的弹力蛋白酶表达是维持正常细胞外基质合成与降解动态平衡的需要;在肝纤维化形成过程中,大鼠胰腺丝氨酸弹力蛋白酶的表达下降是促进肝纤维化形成的因素之一.     

Studies on the Distribution and Metabolism of 14C-dimethylnitrosamine in Foetal and Young Mice

Abstract In pregnant mice injected with ¹⁴C-dimethylnitrosamine, whole-body autoradiography was performed with hemisections at -80° (to prevent evaporation of the volatile dimethylnitrosamine) and with dry tape sections (to localize the non-volatile metabolites). The results indicated that the non-metabolized substance passed to the foetal tissues with a uniform distribution and without formation or accumulation of non-volatile metabolites. Autoradiography in young (1-10 days old) and adult mice showed a high level of metabolites in the liver already 5 min. after the administration of ¹⁴C-dimethylnitrosamine. No metabolism of the substance could be detected at in vitro incubations of liver tissue obtained from foetuses on the last day of gestation (¹⁴CO²-production and incorporation of radioactivity in acid-insoluble macromolecules were used as metabolic indices). However, in vitro experiments with livers of 1-5 days old mice indicated a rapid increase in enzymatic activity after birth. Studies in vivo showed an increased incorporation of radioactivity in the acid-insoluble macromolecules of the liver and a decreased exhalation of ¹⁴CO₂ in 10 and 14 days old mice as compared with 21 and 60 days old mice. This indicates a difference in the fate of dimethylnitrosamine in vivo between the young and older mice.     

大蒜阻断串珠镰刀菌促进二甲基亚硝胺合成的观察

大蒜可抑制致癌性霉菌-串珠镰刀菌的生长及阻断该菌还原NO_3~-产生N0_2~-和促进二甲基亚硝胺合成的作用,为食用大蒜作癌症的病因性预防提供了一定的科学依据。