Hepatocellular carcinoma induces body mass loss in parallel with osmolyte and water retention in rats.

AIMS: The number of cancer survivors with cardiovascular disease is increasing. However, the effect of cancer on body fluid regulation remains to be clarified. In this study, we evaluated body osmolyte and water imbalance in rats with hepatocellular carcinoma. MAIN METHODS: Wistar rats were administered diethylnitrosamine, a carcinogenic drug, to establish liver cancer. We analyzed tissue osmolyte and water content, and their associations with aldosterone secretion. KEY FINDINGS: Hepatocellular carcinoma rats had significantly reduced body mass and the amount of total body sodium, potassium, and water. However, these rats had significantly increased relative tissue sodium, potassium, and water content per tissue dry weight. Furthermore, these changes in sodium and water balance in hepatocellular carcinoma rats were significantly associated with increased 24-h urinary aldosterone excretion. Supplementation with 0.25% salt in drinking water improved body weight reduction associated with sodium and water retention in hepatocellular carcinoma rats, which was suppressed by treatment with spironolactone, a mineralocorticoid receptor antagonist. Additionally, the urea-driven water conservation system was activated in hepatocellular carcinoma rats. SIGNIFICANCE: These findings suggest that hepatocellular carcinoma induces body mass loss in parallel with activation of the water conservation system including aldosterone secretion and urea accumulation to retain osmolyte and water. The osmolyte and water retention at the tissue level may be a causative factor for ascites and edema formation in liver failure rats.

Argininosuccinate synthase as a novel biomarker for inflammatory conditions.

Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.

Carcinoembryonic antigen-related cell-cell adhesion molecule C-CAM is greatly increased in serum and urine of rats with liver diseases.

C-CAM (rat cell CAM/human CD66a) is ubiquitous and multifunctional. It is involved in intercellular adhesion, signal transduction and cell growth inhibition. Structurally, it is related to the carcinoembryonic antigen. In the present study serum, bile and urine of rats with liver diseases were analyzed for the presence of cell CAM. After bile duct ligation and during galactosamine (GalN) hepatitis we found that large amounts of liver membrane-bound C-CAM are secreted or shed into blood. The serum level of another liver membrane-bound protein, LI-cadherin, is not increased. It was shown that C-CAM is also present in bile fluid, and for the first time that C-CAM is present in the urine of rats with liver diseases. A particularly high concentration was measured in the urine of rats suffering from GalN hepatitis.

Inhibition of aflatoxin Ml excretion in rat urine during dietary intervention with oltipraz.

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz) is an effective chemopreventive agent against several classes of carcinogens in many target organs. Induction of carcinogen detoxication enzymes, particularly glutathione S-transferases, appears to be an important component of the protective actions of oltipraz. It has recently been observed that addition of oltipraz to rat liver microsomes or to cultured human hepatocytes blocks the oxidative metabolism of aflatoxin B1 (AFB1) to its 8,9-oxide and the hydroxylated derivative aflatoxin Ml (AFM1). 0ltipraz is a competitive and perhaps irreversible inhibitor of cytochromes P450 1A2 and 3A4. To determine whether oltipraz can affect cytochrome P450-dependent metabolism of AFB1 in vivo we have assessed the effect of oltipraz on the urinary excretion of oxidative metabolites of AFB1 before, during and after a transient intervention. Male F344 rats, housed individually in glass metabolism cages, were gavaged daily with 25 microg [3H]AFB1 for 28 consecutive days. Starting on day 6 and extending to day 16 half of the rats were fed a diet supplemented with 0.075% oltipraz. Sequential 24 h urine samples were collected and a subset analyzed for AFB1 metabolites. AFM1 was the major metabolite detected in all urine samples, accounting for 2-6% of the administered dose. The excretion of AFM1 was greatly reduced (77%) during the active phase of the intervention, when oltipraz was added to the diet, but rapidly returned to control levels after cessation of oltipraz administration. This inhibition of AFM1 excretion was not seen in animals receiving oltipraz by gavage 24 h prior to dosing with AFB1. Collectively these data are consistent with the view that oltipraz or a short-lived metabolite inhibits cytochrome P450 1A2 in vivo.

Urinary and serum mutagenicity studies with rats bearing experimental tumours.

Urine and serum samples of rats bearing three different experimental tumours (hepatocellular carcinoma, myelomonocytic leukemia and mesoblastic nephroma) were investigated for mutagenicity with the Ames Salmonella test. Enhancement of mutagenic activity in TA98 and TA100 was observed only in the case of urine samples obtained from animals bearing nephromas. Mutagenicity increased with increasing time after implantation of tumours. There was no coincidence between urinary and serum mutagenicity under the experimental conditions employed. Further studies are needed to determine the origins, and chemical and genotoxic characteristics of urinary mutagens. In addition, the question as to whether any mutagenic substances can be detected in fractions of plasma/serum should also be experimentally addressed.

Studies on liver tumor promotion in the rat by orotic acid: dose and minimum exposure time required for dietary orotic acid to promote hepatocarcinogenesis.

Our earlier studies indicated that orotic acid, a precursor for pyrimidine nucleotide biosynthesis, exerts a promoting effect on rat hepatocarcinogenesis. The present study was designed to determine the optimum conditions of exposure to orotic acid required for promotion of hepatocarcinogenesis in the initiated rats. The first series of experiments was designed to determine the optimum dose of orotic acid needed to exert its liver tumor promoting effect. Accordingly male Fischer rats were given diethylnitrosamine (200 mg/kg, i.p.) or 0.9% NaCl. One week later carcinogen-injected rats were divided into six groups and fed either basal diet or the same diet containing 0.1, 0.5, 1, 2 or 4% orotic acid. Rats given 0.9% NaCl were fed 4% orotic acid. Two-thirds partial hepatectomy was performed on all animals 10 weeks after starting on their respective diets, and all groups were killed 3 weeks thereafter. Analysis of gamma-glutamyltransferase-positive foci and nodules revealed that 0.5-1% orotic acid in the diet is sufficient to exert a significant promoting effect on the selective growth of initiated hepatocytes, while higher concentrations of orotic acid were only marginally more effective. No gamma-glutamyltransferase-positive foci were observed in animals given 4% orotic acid diet following saline injection. Using 1% orotic acid as the promoting regimen, in the next series, the minimum exposure time required for dietary orotic acid to promote liver carcinogenesis was determined. Male Fischer 344 rats were given i.p. either 1,2-dimethylhydrazine dihydrochloride (100 mg/kg) or 0.9% NaCl 18 h after 2/3 partial hepatectomy. After 1 week of recovery one group of rats was continued on a semisynthetic basal diet, while others were transferred to the same basal diet containing 1% orotic acid. Rats that were on the 1% orotic acid diet were progressively transferred to the basal diet after 5, 10, 20, 29 and 40 weeks of exposure. All rats were sacrificed 54 weeks after the beginning of the experiment. The results indicate that 100% of the initiated rats developed hepatic nodules whether or not they were exposed to an orotic acid-containing diet. However, the incidence of hepatocellular carcinoma was greatly increased in animals exposed to the orotic acid diet, with 42% incidence in initiated rats given orotic acid diet for 10 weeks and up to 75% in those exposed to this diet for 40 weeks. Further, promotion by orotic acid exhibited a high metastatic potential with 33-60% metastasis to the lungs.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular epidemiology of aflatoxin exposures: validation of aflatoxin-N7-guanine levels in urine as a biomarker in experimental rat models and humans.

Human epidemiology and experimental animal data have provided the statistical association and biological information necessary to propose that aflatoxins are risk factors for human liver cancer. As liver cancer causes at least 200,000 deaths per year, prevention measures must be developed to ameliorate this nearly always fatal disease. Preventive strategies will be facilitated by the identification of individuals at high risk. It is the goal of the molecular dosimetry field to provide facile and accurate biomarkers to identify people at high risk for carcinogen exposure and consequent adverse health effects. We have developed methods to defect the major aflatoxin DNA adduct, aflatoxin N7-guanine (AFB-N7-guanine), in urine, examined the dose-response characteristics in people living in China and The Gambia, and have found an excellent association of this biomarker with exposure. In addition to exposure studies in people, our laboratories have monitored AFB-N7-guanine excretion in the urine of rats whose risk for developing cancer has been modulated with dietary chemoprotective agents such that independent groups of animals receiving the same dosage of aflatoxin B1 were at either high or low risk for tumorigenesis. The production of DNA damage by aflatoxins is not the exclusive mechanism for liver cancer. Many other factors, including hepatitis B virus, cell proliferation, and nutritional status, can exert strong modification effects in human disease. Thus, molecular epidemiological investigations that examine only one biomarker may greatly underestimate or overestimate the risk for an individual.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermogenesis, low-protein diets, and decreased development of AFB1-induced preneoplastic foci in rat liver.

The development of hepatocellular, putatively preneoplastic, gamma-glutamyl transpeptidase positive (GGT+) foci and tumors induced by aflatoxin B1 (AFB1) has been shown to be reduced in male F344 rats fed a diet containing 6% protein (as casein). This reduction occurs despite increased energy intake, when compared with animals fed a diet containing 22% protein. Among its many effects, low protein intake is known to increase the proportion of energy intake expended in the form of heat (thermogenesis); thus, this investigation examined the association between the development of GGT+ foci and alterations in indices of thermogenesis induced by feeding varying levels of dietary protein. Five days following the completion of AFB1 dosing, animals were assigned to groups fed 4%, 8%, 12%, 16%, or 22% dietary protein for 6 weeks. Foci development (% liver volume occupied) was markedly reduced in animals fed the low-protein diet (4%, 8%), yet calorie consumption per 100 g body wt was greater. A modest negative linear trend was observed in oxygen consumption with increasing levels of dietary protein intake. Urinary norepinephrine levels were elevated in the groups fed 4% and 8% protein; urinary dopamine and norepinephrine turnover rates in brown adipose tissue were highest in animals fed 4% protein. These results suggest that GGT+ foci development occurs when a "critical level" (approx 12%) of dietary protein intake is reached. Inhibition of foci development at lower levels of protein intake is associated with several indicators of increased thermogenesis.

Polyamine metabolism and polyamine excretion in normal and tumor bearing rodents.

Aminoguanidine sulfate (AG) inhibits in vivo oxidative deaminations of the polyamines and their derivatives. This compound was used to study urinary polyamine excretion by normal, and tumor bearing rodents. Of the total expendable polyamines, 64 percent were catabolized by AG-sensitive oxidases and escaped observation. Tumor bearing animals did not excrete enhanced amounts of polyamines at any stage of tumoral growth. However, treatment with adriamycin caused an increased polyamine excretion. Prolonged administration of a 2% solution of a-difluoromethylornithine (DFMO), reduced urinary polyamine excretion to the same level of about 27%, irrespective whether the animals carried a large tumor or not. Cadaverine excretion was not affected by treatment with DFMO. Based on these animal data, it appears that urinary polyamines are of restricted value in the diagnosis of tumors.

Urinary catabolites of ribonucleic acid as cancer markers: a preliminary report of their use in patients with lung cancer.

Rats with aflatoxin-B1-induced hepatomas and dimethylnitrosamine-induced nephroblastomas excreted greater than normal amounts of urinary modified nucleosides and bases, catabolites of ribonucleic acid (RNA). Although both neoplasms caused increased excretions of the same catabolites, their quantitative profiles differed, suggesting that it may be possible to distinguish between tumors. Rats with transplanted tumors (e.g., hepatomas and osteogenic sarcomas) did not excrete elevated levels of urinary RNA catabolites until approximately 20 days after transplantation despite rapid growth of the tumor for the first 15 days. These data suggest that the source of the elevated levels of these excretory products may be the host's tissue RNA. Preliminary studies in human beings with lung cancer showed marked elevation of one or more urinary RNA catabolites. Resection of the diseased tissue in 2 patients caused a drop in levels. The measurement of urinary RNA catabolites may be useful in the diagnosis, prognosis, and evaluation of therapy in patients with lung cancer.

Excretion of RNA catabolites by rats with hepatoma transplants.

Rats with transplants of Morris Hepatoma 5123 excreted in their urine greater than normal amounts of modified nucleosides and bases, catabolites of RNA. Despite rapid growth of the neoplasm, the elevated levels did not appear until 22 days after inoculation with the tumor. With tumor progression, there were increased levels and number of these catabolites. This study also suggests that the source of the elevated RNA catabolites is mainly from the host RNA rather than from tumor tissue and also that mRNA and rRNA as well as tRNA may contribute to the urinary levels.

Comparison of urinary modified nucleosides and bases in rats with hepatomas and nephroblastomas.

Hepatomas were induced in rats with aflatoxin B1, and nephroblastomas with dimethylnitrosamine. Microscopic examination of livers of aflatoxin-treated rats revealed multinodular hepatocyte hyperplasia at 8 months, and by 13 months all rats had hepatomas. Nephroblastomas were observed by 4 months and by 8 months all rats had developed them. The urinary excretion of several modified nucleosides and bases by normal rats is dependent on body weight and reflects, to a certain extent, their concentrations in tissue tRNA. Increased levels of several modified nucleosides and bases were found in all rats that had cancer. Rats with hepatomas excreted essentially the same modified nucleosides and bases as did those with nephroblastomas; the quantitative patterns of excretion were different, however, suggesting that the urinary modified nucleosides and bases may be used to differentiate between neoplasms. Although the increase in urinary modified nucleosides and bases by tumor-bearing animals results primarily from more rapid turnover of neoplastic tRNAs, the data indicate that increased turnover of mRNA and possibly rRNA may occur in neoplastic tissue. Preliminary data suggest that increases in urinary modified nucleosides and bases may occur during a precancerous stage. The urinary pattern of modified nucleosides and bases by rats with hepatomas is altered if another primary tumor is present. The results obtained from these studies support the use of modified nucleosides and bases in urine as biochemical markers of cancer.

Enhanced urinary excretion of N1-acetylspermidine and the presence of tumors.

We have studied the urinary excretion of free and acetylated polyamines in hepatoma-bearing Buffalo rats during the period of linear growth of the tumor mass. The excretion of nonconjugated polyamines was unchanged. N1-Acetylspermidine excretion did not parallel the linear increase in tumor mass but increased exponentially. Enhancement of N8-acetylspermidine excretion above control levels was observed only at a time when the average tumor mass was 35 +/- 9 (S.D.) g. shortly before the period when necrosis is usually observed. These data taken together with the analysis of urinary acetylpolyamines in rats bearing mammary tumors and in two melanoma patients show that the determination of the N1-acetylspermidine/N8-acetylspermidine ratio in urine may be of only limited value as an indicator for the presence of tumors.

Tissue and urinary glycosaminoglycan patterns associated with a fast, an intermediate, and a slow-growing Morris hepatoma.

The purpose of this investigation was to evaluate the glycosaminoglycans (GAG's) in different behavioral-histological types of i.m.-transplanted hepatomas and in the liver and urine of animals bearing these tumors. Groups of 10 Buffalo rats carrying fast-growing (7777), intermediate (5123tc), and slow-growing (9618A) Morris hepatomas were studied as the tumors reached 3 cm. Urinary and tissue GAG's were isolated by proteolysis, separated as cetylpyridinium complexes, and measured as uronic acid. The GAG's were further purified using anion-exchange chromatography and characterized with mucopolysaccharidases. Tissue GAG,s were also evaluated histochemically using Alcian blue staining and mucopolysaccharidases. Tissue from fast-growing, intermediate, and slow-growing tumors exhibited greater GAG levels than did normal liver in the hyaluronic acid (0.4 M NaCl-soluble) fraction and in the chondroitin sulfate-heparan sulfate (1.2 M NaCl-soluble) fraction. The livers of tumor-bearing animals exhibited GAG levels similar to those of normal liver. Increased urinary GAG excretion was appreciated in animals bearing Tumors 5123tc and 9618A but not in those bearing Tumor 7777.