Orotic acid, a new promoter for experimental liver carcinogenesis

Male Fischer 344 rats initiated with 1,2-dimethylhydrazine 2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to a diet containing 1% orotic acid for 13 months developed a 100% incidence of hepatocellular carcinoma. The creation of nucleotide pool imbalances by dietary orotic acid, for e.g., an increase in uridine nucleotides and a decrease in adenine nucleotides, was considered as a possible mechanism for the promotional effect of orotic acid on liver carcinogenesis. The significance of this hypothesis is that altered nucleotide pools affect both genomic as well as membrane organization. Consistent with this hypothesis is our finding that feeding rats with a diet containing 1% orotic acid for 10 weeks resulted in a liver DNA damage as monitored by its slower sedimentation in alkaline sucrose gradients compared to the corresponding controls. To assess the general applicability of this hypothesis, nucleotide pool imbalances were created by using methods other than feeding orotic acid and their effect on the incidence of gamma-glutamyltransferase positive foci in carcinogen initiated rats was determined. The results obtained indicated that rats initiated with 1,2-dimethylhydrazine.2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to diet deficient in arginine, a regimen that causes an increased synthesis and excretion of orotic acid, or were fed diets containing 1% thymidine or 1% thymine developed greater number of gamma-glutamyltransferase positive foci compared to the corresponding controls fed the basal diets. These results were interpreted to indicate that orotic acid exerts its promotional effect probably by creating an imbalance in nucleotide pools. One of the mechanisms by which an imbalance of nucleotide pools influences the pathogenesis of the carcinogenic process may be by inducing perturbations in the DNA.     

Dose response study of N-nitrosodiethanolamine initiation of rat hepatocarcinogenesis.

Initiating activity of N-nitrosodiethanolamine (NDELA) for rat liver carcinogenesis was investigated using an 8-weeks bioassay system. Male F344 rats were initially treated with a single intraperitoneal injection of NDELA at one of five dose levels: 1,600, 800, 400, 200, or 100 mg/kg. Two weeks later, the rats were placed on 0.02% 2-acetylaminofluorene (2-AAF) or 0.05% phenobarbital (PB) containing diet for 6 weeks. All animals were subjected to 2/3 partial hepatectomy 4 weeks after the NDELA treatment, and killed at the end of the eighth week. NDELA itself exerted low toxicity in terms of body weight gain. Clear dose-dependent initiating activity of NDELA was observed in terms of development of glutathione S-transferase placental form (GST-P) positive liver cell foci, this being more apparent with PB promotion than with 2-AAF where the enhancing regimen itself caused multiple lesion development. Initiating potential of NDELA, however, was much lower than that observed for diethylnitrosamine in our previous work.     

Possible prevention by abieslactone of development of diethylnitrosamine-initiated GST-P positive foci in the rat liver

Triterpenoid compounds, isolated from plants of Abies genus (Pinceae), are known to exert anti-tumor promotion activities in mouse skin carcinogenesis. In the present study, we investigated whether AVB-1 and acid and acid methyl ester derivatives have inhibitory effects on rat hepatocarcinogenesis by using a liver medium-term bioassay for carcinogens (Ito's test), immunohistochemically assessing the numbers and areas per cm(2) of preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci. In experiment 1, 6-week-old male Fisher 344 rats were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) and subjected to two-thirds partial hepatectomy at week 3. From weeks 2 to 8, the compounds were given three times a week at a dose of 1 mg/kg b.w. by i.g. gavage and found to significantly decrease the number of GST-P-positive foci in the liver. In experiment 2, AVB-1 was given three times a week at doses of 3, 1, or 0.3 mg/kg b.w. by i.g. gavage from weeks 2 to 8. All doses of AVB-1 significantly decreased the numbers of GST-P-positive foci. Thus, our results suggest that AVB-1 is a chemopreventive agent for rat hepatocarcinogenesis.     

Complementarity between two rat liver tumor promoters

A delay in the exposure of initiated rats to orotic acid (OA) beyond a specific time frame results in a progressive loss of promotional effect in liver carcinogenesis. The current study was designed to ascertain whether the loss of promotional effect could be counteracted by pre-exposing the initiated animals to other rat liver promoting regimens such as a diet deficient in choline (CD). Male Fischer 344 rats (150 g) were initiated with diethylnitrosamine (200 mg/kg, ip); 1 week later they were given either a CD diet or a CD diet supplemented with choline for 5 weeks. Animals from these two groups were then fed either a 1% OA diet or the basal diet for another 20 weeks. The results indicated that the loss of OAs promotion efficacy from delaying the start of the promoting regimen can be counteracted by pre-exposing the initiated rats to a CD diet. Thus in rats exposed to OA from the first week of initiation, 7% of the liver developed as nodular areas, whereas only 0.8% of liver was nodular when OA feeding was delayed by 5 weeks. This loss was abolished when initiated rats were fed a CD diet for 5 weeks prior to feeding OA for 20 weeks. These results suggest that in a rat liver tumor promotion model, two tumor promoters, OA and CD, show some degree of complementarity when given sequentially.     

Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern.

As demonstrated previously, liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-diabetic rats are altered in various respects. The hepatocytes in these acini store glycogen and/or fat, and they show an increase in proliferation as well as in apoptotic activity. Thus, they are phenotypically similar to carcinogen-induced preneoplastic liver foci (glycogen-storing foci and sometimes also mixed cell foci). By means of catalytic enzyme histochemistry or immunohistochemistry, we investigated the activity of key enzymes of alternative pathways of carbohydrate metabolism and some additional marker enzymes (well known from studies on preneoplastic hepatic foci) in the altered liver acini surrounding the islet isografts. In addition, the expression of glucose transporter proteins 1 and 2 (GLUT-1 and GLUT-2) were investigated immunohistochemically. The activities of hexokinase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and glucose-6-phosphate dehydrogenase were increased, whereas the activities of glycogen phosphorylase, adenylate cyclase, glucose-6-phosphatase, and membrane-bound adenosine triphosphatase were decreased in the altered liver acini. The expression of GLUT-2 was also decreased. GLUT-1 and glutathione S-transferase placental form were not expressed, and the activities of glycogen synthase and gamma-glutamyl-transferase remained unchanged. All changes of the enzyme activities were in line with the well known effects of insulin and resembled alterations characteristic of preneoplastic liver foci observed in different models of hepatocarcinogenesis. It remains to be clarified in long-term experiments whether or not these foci represent preneoplastic lesions and may proceed to neoplasia.     

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.     

Effects of fasting and intermittent fasting on rat hepatocarcinogenesis induced by diethylnitrosamine

The influences of fasting on DEN-initiation and of intermittent fasting (IF) on the rat liver chemical carcinogenesis process were evaluated in a 52-week long assay. Three groups of adult male Wistar rats were used: Groups 1 to 3 were treated with a single i.p. injection of 200 mg/kg of diethylnitrosamine (DEN). Group 2 was submitted to 48 h fasting prior to DEN treatment. After the 4th week, Group 3 was submitted to IF, established as 48 h weekly fasting during 48 weeks, while Groups 1 and 2 were fed ad libitum until the 52nd week. All animals were submitted to 70% partial hepatectomy and sacrificed at the 3rd and 52nd weeks, respectively. Fasting prior to DEN-initiation did not influence the development of altered foci of hepatocytes (AFHs) and of hepatic nodules (Group 2 vs. Group G1). IF inhibited the development of preneoplastic lesions, since this dietary regimen decreased the number and the size of glutathione S-transferase (GST-P) positive foci and the number and size of liver nodules (Group G3 vs. Group G1). The inhibitory effect of IF was also reflected in the development of clear and basophilic cell foci. These results indicate that long-term IF regimen exerts an anti-promoting effect on rat hepatocarcinogenesis induced by DEN.     

Further studies on the tumor-initiating activity of the beta-blocker DL-ZAMI 1305

The purpose of this study was to evaluate the initiating activity of the hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) by the initiation-promotion protocol of Pereira. Female Wistar rats were given a single dose 150 mg/kg of body weight of DL-ZAMI 1305 by gavage 24 hours before or 24 hours after partial hepatectomy. One week later rats were given phenobarbital (0.05%) in the diet for a period of 7 weeks. DL-ZAMI 1305-treatment resulted in the appearance of gamma-glutamyltranspeptidase foci and of other preneoplastic lesions in all animals. Preneoplastic lesions were also present in a fraction of DL-ZAMI 1305-treated animals not subjected to partial hepatectomy, whether given or not phenobarbital. Results obtained in a separate experiment demonstrated that DL-ZAMI 1305-treatment inhibits cell proliferation and induces DNA damage in the regenerating rat liver. The results of this study clearly demonstrated that the beta-blocker DL-ZAMI 1305 is an initiating carcinogen for the liver of female Wistar rats.     

Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens

Our laboratory has developed an in vivo short-term screening test for hepatocarcinogens based on quantitation of gamma-glutamyl transpeptidase (gamma-GT) positive foci. However, gamma-GT positive hepatocytes appear in periportal areas under a variety of circumstances apparently unrelated to hepatocarcinogenesis. Glutathione S-transferase placental type (GST-P), which is hardly detectable in normal rat liver, was recently demonstrated as a new marker protein for preneoplastic liver foci. In experiment I, rats were initially given a single dose (200 mg/kg) of diethylnitrosamine intraperitoneally and 2 weeks later were treated with test compounds for 6 weeks. All rats were subjected to partial hepatectomy at week 3. The long-term development of preneoplastic lesions was followed in rats for 50 weeks. The immunohistochemical investigation of GST-P binding and the histochemical demonstration of gamma-GT in serial sections revealed that almost all gamma-GT foci were GST-P positive, but 5-10% of GST-P foci could not be detected by gamma-GT staining. From week 8, many gamma-GT foci partially lost gamma-GT activity. However, no comparable disappearance of GST-P was evident in the lesions. All hepatocellular carcinomas (HC) found at week 50 consisted of GST-P positive HC cells. In contrast, 37.9% (11/29) of HC were negative for gamma-GT. In experiment II (in vivo short-term screening test for hepatocarcinogens), rats were treated in the same manner as for experiment I and killed at week 8. Fifty-eight chemicals were investigated for their potential to modify GST-P positive foci development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocellular neoplasms induced by low-number pancreatic islet transplants in streptozotocin diabetic rats.

We have previously demonstrated in short-term experiments that altered hepatocytes in liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-induced diabetic rats with mild persisting diabetes resemble those in preneoplastic foci of altered hepatocytes. We now present the results of long-term studies (up to 22 months) in this animal model. Glycogen-storing foci (which were the first parenchymal alteration observed some days after transplantation) persisted at least for 6 months, when the first mixed-cell foci and the first hepatocellular adenoma emerged. After 15 to 22 months, 86% of the animals exhibited at least one hepatocellular adenoma and four animals (19%) showed a hepatocellular carcinoma. The transplants were found in a close spatial relationship with the preneoplastic foci and the hepatocellular neoplasms. The mitotic indices, the 5-bromo-2'-desoxyuridine labeling indices and the apoptotic indices showed significant differences between the unaltered liver parenchyma, different types of preneoplastic foci, and hepatocellular neoplasms. The immunohistochemical expression of transforming growth factor-alpha increased during the stepwise development from glycogen-storing liver acini to hepatocellular carcinomas. Hepatocarcinogenesis in this new animal model is probably due to the hormonal and growth-stimulating effects of insulin secreted by the intraportally transplanted islets of Langerhans in diabetic rats.     

Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.     

The variations of S-adenosyl-L-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis

A decrease in liver S-adenosyl-L-methionine (SAM) content and an increase in ornithine decarboxylase (ODC) activity occurred between the 2nd and the 5th week after starting 2-acetylaminofluorene (AAF) feeding in diethylnitrosamine (DENA)-initiated rats. These rats then received a 0.05% phenobarbital (PB)-containing diet for 18 weeks after the end of AAF feeding. Two weeks after starting AAF, an increase in the hepatocyte labeling index (LI) also occurred in gamma-glutamyl-transpeptidase (GGT)-positive foci and surrounding tissue. LI returned to control values in a few days in surrounding tissue, while it remained high for at least 4 weeks in the foci. Analogous changes were observed, but for a shorter period of time, in the rats subjected to partial hepatectomy (PH) plus AAF, in which no GGT-positive foci developed. Twenty-four weeks after starting AAF, 30% of the liver was occupied by visible nodules. ODC activity and LI were high and SAM was low in nodules, but they were near to control values in surrounding liver. SAM administration reconstituted the liver SAM pool, inhibited ODC activity, and prevented visible nodule development. SAM inhibition of ODC activity occurred in vitro only after preincubation with liver homogenate and was enhanced by adenine, an inhibitor of methylthioadenosine (MTA) phosphorylase. MTA addition to the reaction of mixture for ODC determination was inhibitory. The SAM decrease in both liver and nodules was coupled with a decrease of MTA content. SAM administration caused MTA accumulation in the liver. It is suggested that liver SAM content by influencing MTA level, could be a rate-limiting factor for growth and promotion, through a modulation of polyamine synthesis.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.     

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)⁺ liver cell foci and the numbers of Ki-67⁺ proliferating cells within GST-P⁺ foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P⁺ foci (area) to 48.9–49.4% and Ki-67⁺ cells to 55.5–61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN^– foci, phospho-Akt substrate⁺ foci, phospho-Smad3^– foci and Smad4^– foci in GST-P⁺ foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN^– foci in GST-P⁺ foci to 59.8–72.2% and Smad4^– foci to 62.4–71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate⁺ foci in GST-P⁺ foci to 75.2–75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.     

The enhancing effect of ethanol on the development of glutatione S-transferase placental form-positive foci induced by diethylnitrosamine in F344 rat

The effects of ethyl alcohol and pig serum administration on the development of preneoplastic hepatic enzyme-altered foci were examined in an in vivo mid-term assay system. Rats were initially given a single dose (200 mg/Kg) intraperitoneal injection of diethylnitrosamine (DEN). Two weeks later, treatment was started with 10% ethanol + 10% sucrose solution, 10% sucrose solution, or tap water as drinking water for 6 weeks with or without intraperitoneal injection of porcine serum twice a week. All rats were subjected to a two-thirds partial hepatectomy at week 3. The modification potentials were evaluated by comparing the number and area per cm2 of glutathione S-transferase placental form-positive (GST-P+) foci in the liver of each group. As a result, ethanol significantly enhanced the development of GST-P+ foci. Unfortunately, the porcine serum injection produced no hepatic fibrosis and no significant alteration in GST-P+ foci.     

Dietary and metabolic manipulations of the carcinogenic process: role of nucleotide pool imbalances in carcinogenesis

Perturbations in DNA and/or membranes are considered to be important for the carcinogenic process. A search for nutritional and metabolic means of disturbing the homeostasis of DNA and membranes revealed that nucleotide pools offer an exciting possibility. An imbalance in nucleotide pools can exert a two-pronged attack on both DNA and membranes. When given to rats, orotic acid, a precursor of pyrimidine nucleotides, results in an imbalance in nucleotide pools (an increase in uridine nucleotides and a decrease in inosine/adenine nucleotides), alterations in both DNA and membranes, and promotion of carcinogenesis in the liver initiated by chemical carcinogens. Agents such as adenine and allopurinol, which inhibit the metabolism of orotic acid and thereby decrease the formation of uridine nucleotides, and galactosamine, which traps uridine nucleotides, inhibited the promotional effects of orotic acid in the liver. These results suggested that orotic acid needs to be metabolized to uridine nucleotides and the creation of a subsequent imbalance in nucleotide pools is important for the promotional effects of orotic acid. To determine whether the creation of a nucleotide pool imbalance is a more general mechanism of tumor promotion, two lines of approach were investigated. One was to determine the effect of orotic acid on promotion of carcinogenesis in other organs, and the second approach was to determine how to induce nucleotide pool imbalances by means other than orotic acid administration. It is interesting to note that orotic acid promotes carcinogenesis in duodenum initiated by azoxymethane. Regarding the second approach, it became apparent that several metabolic disturbances result in increased orotic acid synthesis and alterations in nucleotide pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠再生肝对二乙基亚硝胺启动作用的敏感性

目的比较再生肝和正常肝对二乙基亚硝胺（ＤＥＮ）启动作用的敏感性。方法以２／３肝叶切除后８周末的大鼠为实验组,正常大鼠为对照组,作如下比较：肝重、常规组织学检查及３Ｈ－ＴｄＲ掺入试验;用修改的Ｓｏｌｔ－Ｆａｒｂｅｒ模型,通过对ＧＧＴ阳性癌前病灶的体视学测量,观察肝脏对ＤＥＮ的启动效应;在体内和体外（无血清原代培养肝细胞）经ＤＥＮ攻击后,以核酸原位缺口标记方法观察肝细胞ＤＮＡ的损伤程度。结果２／３肝叶切除后８周末的实验组肝脏的修复过程已完成,未见肝细胞继续增生的表现;经ＤＥＮ攻击后,实验组肝癌前病灶在数密度和体积密度上都显著高于对照组;无论在体内或体外接受ＤＥＮ攻击后,实验组肝细胞ＤＮＡ的损伤程度都显著大于对照组。结论即使再生过程已经完成,再生肝仍比正常肝具有较高的致癌敏感性,这与再生肝肝细胞在ＤＥＮ攻击后其ＤＮＡ损伤较重相关。     

Involvement of PTEN/Akt signaling and oxidative stress on indole-3-carbinol (I3C)-induced hepatocarcinogenesis in rats

We previously reported that indole-3-carbinol (I3C) had hepatocellular tumor-promoting activity in a short-term (8 weeks) two-stage liver carcinogenesis model in rats. It was suggested that this effect was related to the production of reactive oxygen species (ROS) caused by cytochrome P450 1A (CYP1A) induction. In the present study, 0.5% I3C was administered to DEN-initiated rats for 26 weeks to examine the effect of prolonged administration of I3C and to clarify the possible mechanisms of I3C-induced hepatocarcinogenesis. The number and area of GST-P positive foci, ROS production, TBARS level, 8-OHdG content and mRNA levels of Ahr and Nrf2 gene batteries significantly increased in the DEN-I3C group compared with the DEN-alone group. Furthermore, some GST-P positive preneoplastic foci progressed to hepatocellular adenomas with the prolongation of I3C administration. Lack of PTEN and phospho-Smad2/3 expression and translocations of PDPK1 and phospho-Akt substrates to underneath the cell membrane were observed in the majority of hepatocellular adenomas. In addition, the number of Ki-67 positive cells increased in adenomas compared with the preneoplastic foci. These results suggest that the administration of I3C for 26 weeks in DEN-initiated rats induces tumor progression from hepatocellular altered foci to hepatocellular adenomas by ROS-mediated Akt activation that inhibits the TGF-β/Smad signaling and results in the increased cell proliferation.     

Hyperproliferative hepatocellular alterations after intraportal transplantation of thyroid follicles

The thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis.     

Neuron tolerance during hydrocephalus.

Whether or not neuron death plays a major role in pathophysiology during hydrocephalus is not well known. The goals of this study were to determine if neural degeneration occurred during hydrocephalus, and to determine if neuron tolerance developed during this pathophysiologic procedure.Neural damage as visualized by a sensitive staining technique, silver impregnation, was observed in three experimental groups: (1) adult hydrocephalic rats induced by kaolin injection into the cisterna magna, (2) adult rats with chronic hydrocephalus for 10 weeks subjected to acute forebrain ischemia induced by four-vessel occlusion, and (3) adult rats without hydrocephalus subjected to acute forebrain ischemia. The magnitude of hydrocephalus was also evaluated during this time. In mild or moderate hydrocephalus, little cell death was found. In severe hydrocephalus, axon and neuropil degeneration was extensively distributed, but cell death was still rarely observed. Although some neuron degeneration was found after acute forebrain ischemia in hydrocephalic rats, the extensive cell death in cortical layers III and V, and in hippocampal areas CA1 and CA4 that is commonly observed in the ischemic brain without hydrocephalus, was not seen.This study suggests that neuron death was not a major pathological change in the brain during hydrocephalus, with cerebral ventricles being enlarged during the development of hydrocephalus. Less neuron death in hydrocephalic rats after acute forebrain ischemia suggests that neuronal tolerance to ischemia occurs during hydrocephalus.