Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.

Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen DL-ethionine.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyltranspeptidase-positive hepatocytes in the liver of carcinogen-treated rats.

The effect of feeding phenobarbital (PHB) with a choline-devoid (CD) diet on the emergence of foci of gamma-glutamyltranspeptidase (GGT)-positive hepatocytes in the liver of carcinogen-treated rats was investigated. Male Sprague-Dawley rats were given a single dose of diethylnitrosamine (50 mg/kg) 18 hr after a partial hepatectomy and 10 days later were placed on a plain choline-supplemented (CS) diet, a plain CD diet, or the CS and CD diets containing 0.06% PHB. Groups of rats were killed after 5 and 7 weeks of feeding each of the four diets, the livers were taken, and the number and size of foci of GGT-positive hepatocytes were determined. In rats fed the CS + PHB diet, the number of foci per sq cm of liver section was greater than that in rats fed the plain CS diet but smaller than that in rats fed the plain CD diet. Addition of PHB to the CD diet resulted in twice as many foci as in the plain CD diet and foci larger than those resulting from the plain CD diet. THe hepatocytes in the foci of rats fed th CD and CD + PHB diets showed, uniformly, not only GGT positively but also a relative absence of fatty change. The results indicate that PHB and a CD diet, when combined, have a synergistic effect in promoting the evolution of liver cells, initiated by a chemical carcinogen, to foci of altered GGT-positive hepatocytes. This promoting regimen may become useful in studies concerned with the initiation and promotion stages of liver carcinogenesis.

Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.

The effects of feeding a choline-deficient (CD) or a choline-supplemented diet upon the early stages of DL-ethionine carcinogenesis in rat liver were investigated. Low levels of DL-ethionine (0.05 and 0.10%) when fed with a CD diet were found to induce within 4 weeks a massive proliferation of oval cells without significant cell necrosis or presence of inflammatory cell infiltrates. The same levels of ethionine when fed with a choline-supplemented diet caused no significant histological alteration of the liver. In rats fed the CD plus ethionine diets concomitant with the proliferation of oval cells, there was a marked elevation in the content of alpha1-fetoprotein in both liver and plasma. After specific immunofluorescence staining, oval cells stained intensely for albumin and alpha1-fetoprotein. Hepatocytes stained only for albumin, and bile duct cells stained for neither albumin nor alpha1-fetoprotein. These results indicate that a diet deficient in choline markedly alters the response of rat liver to carcinogenetic doses of ethionine. Thus, ethionine hepatocarcinogenesis in rats fed a CD diet may be a useful model for the exploration of the mechanism(s) whereby a dietary factor influences hepatocarcinogenesis.

Stimulation of DNA synthesis and cell proliferation in the liver of rats fed a choline-devoid diet and their suppression by phenobarbital.

Feeding of choline-devoid (CD) diet and dietary administration of phenobarbital (PHB) are efficient promoters of liver carcinogenesis in the rat. Furthermore, inclusion of PHB in a CD diet results in a synergistic effect, inasmuch as the promoting action of their combination is greater than the sum of those exerted by either agent alone. To investigate the mechanism(s) of action of the two promoters, liver DNA synthesis and liver cell proliferation were studied in rats fed a CD diet, a choline-supplemented diet, or the same diets to which 0.06% PHB was added. DNA synthesis was determined by [3H]thymidine incorporation into DNA and autoradiography, and cell proliferation was determined by mitosis counts. Feeding the CD diet caused an increase of both DBA synthesis and cell proliferation over those present in rats fed the choline-supplemented diet. Inclusion of PHB in the CD diet, on the other hand, inhibited DNA synthesis and cell proliferation. These results indicate that stimulation of cell proliferation per se may not be a sufficient condition for an agent to act as a promoter of liver carcinogenesis.

Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat.

Male Fisher 344 rats were solely fed a choline-supplemented diet for 65 to 105 weeks or a choline-devoid diet for 24 to 102 weeks. Hepatocellular carcinomas developed in the latter animals, beginning at 24 weeks. Other groups of rats were given a single dose of 20 mg diethylnitrosamine/kg, 18 h after a partial hepatectomy and were fed, 4 weeks thereafter, either a choline-supplemented, or a choline-devoid diet for up to 48 weeks. In rats fed the choline-supplemented diet, the only relevant lesion observed was a small transect number of foci of enzyme-altered hepatocytes. On the other hand, a significant number of foci, of preneoplastic nodules, and of hepatocellular tumors developed in rats fed the choline-devoid diet. The results obtained are consistent with those previously reported by others, indicating that diets devoid of choline, or of choline and methionine, are carcinogenic. The diets appear to act as complete carcinogens, since they are also efficient promoters of chemical hepatocarcinogenesis, as shown again, in the present study, by the results obtained in the diethylnitrosamine-pretreated rats.

Persistent reduction of indigenous DNA modification (I-compound) levels in liver DNA from male Fischer rats fed choline-devoid diet and in DNA of resulting neoplasms.

Reduced levels of putative indigenous DNA modifications (I-compounds) in liver DNA of male Fischer 344 rats fed a hepatocarcinogenic choline-devoid (CD) diet for up to 7 mo have been previously reported. To investigate the persistence of this effect and possible relationships between I-compounds and hepatocarcinogenesis, liver DNA modifications of tumor-free male rats fed a CD diet for 3, 6, 9, or 12 mo, followed by a choline-supplemented (CS) diet to 16 mo, were compared with those in rats fed exclusively the CD or CS diet for 16 mo by a 32P-postlabeling assay. In addition, DNA from nontumorous and tumorous tissues of rats fed the CD diet similarly for 12 or 16 mo was analyzed. It was found that total I-compound levels in male rats consecutively fed CD and CS diets for various lengths of time were similar to those in rats fed the CD diet only and significantly lower than those in rats fed the CS diet only. I-compound levels of nontumorous regions from tumor-bearing livers were 73% of those in tumor-free livers from the same treatment group. I-compound levels were further reduced, some to undetectable levels, in tumor tissues and exhibited an inverse relationship with tumor incidence. The patterns and levels of I-compounds in liver DNA of CD diet-fed female rats, which were not susceptible to CD diet-induced hepatocarcinogenesis, on the other hand, were not significantly different from those of controls. Thus, reduction of I-compound levels by feeding a CD diet lasted for many months after changing from the CD to the CS diet. Whether this persistent DNA alteration contributes to carcinogenesis remains to be determined.

Isolation and characterization of nascent albuminyl peptides synthesized in vivo from rat liver.

A procedure is described for the isolation of nascent albuminyl peptides from rat liver polysomes which is based on the isolation of total peptidyl tRNA by ion-exchange chromatography on ECTEOLA cellulose followed by immuno-affinity chromatography employing monospecific anti-albumin antibodies immobilized on Sepharose 4B. Identity of the isolated nascent albuminyl peptides was assayed by tryptic peptide fingerprint analysis. Quantitation and determination of the specific activity of the nascent albuminyl peptides, labeled in vivo with l-[14c]leucine, were made by subjecting the peptides to acid hydrolysis, dansylation and resoultion of the amino acids by thin-layer chromatography, and determination of the specific activity of dansyl leucine.

Reduced response to phenobarbital by the liver of rats fed a choline-deficient diet.

1. The response of the liver to phenobarbital administration was compared in rats fed either laboratory chow, a semipurified choline-supplemented diet or a semipurified choline-deficient diet. 2. The liver contents of proteins, lipids and cytochrome P-450, as well as the activity of aminopyrine and ethylmorphine demethylases, were measured after 5 days of feeding and five daily injections of phenobarbital. Liver sections were examined electron microscopically. 3. In rats fed the choline-supplemented diet, phenobarbital administration caused increases in cellular constituents, enzyme activities and smooth endoplasmic reticulum membranes as great as those seen in rats fed laboratory chow. However, in rats fed the choline-deficient diet, the response of the liver to phenobarbital administration was severely reduced in comparison to that in rats fed the other diets. 4. It is concluded that dietary choline and an adequate synthesis of lecithins are necessary for the induction of microsomal mixed-function oxidases and the concomitant accumulation of smooth endoplasmic reticulum in hepatocytes.

The PLAT Study: a multidisciplinary study of hemostatic function and conventional risk factors in vascular disease patients.

In this paper are reported the basal results of a multidisciplinary, multicenter study designed to explore in a population with ischemic disease the relation between hemostatic variables, conventional risk factors and atherothrombotic sequelae. 953 patients less than or equal to 69 yrs with documented coronary, cerebral or peripheral atherosclerotic disease were studied and followed-up for 24 months. Examinations included hemostatic and lipid laboratory assays, arterial Doppler examination, cerebral computerized tomography and nuclear magnetic resonance, exercise electrocardiogram and coronary angiography. Fibrinogen (301.4 +/- 71.52 mg/dl) correlated positively with antithrombin III (r = 0.27) and leukocytes (r = 0.25), negatively with HDL-cholesterol (r = 0.18) and tended to increase with smoking. Heavy smokers had higher leukocyte counts than non-smokers (8.0 +/- 2.0 vs. 7.2 +/- 2.1 x 10(3)/microliters), higher triglycerides (1.87 +/- 1.12 vs. 1.53 +/- 1.35 mmol/l) and lower HDL-cholesterol (0.93 +/- 0.27 vs. 1.00 +/- 0.25 mmol/l). FVII correlated positively with triglycerides (r = 0.16) and protein C (r = 0.45). vWF:Ag (145.4 +/- 70.58%) ad FVII:C (139.7 +/- 59.10%) were positively correlated (r = 0.44). FVIII:C correlated positively with fibrinogen (r = 0.21). Myocardial infarction survivors with associated cerebral and peripheral vascular lesions had higher FVIII:C, FVII, fibronogen and vWF:Ag. These findings suggest that hemostatic factors may enhance and/or mediate the effects of conventional risk factors in atherothrombotic ischemic events.

Association of increased fibrin turnover and defective fibrinolytic capacity with leg atherosclerosis. The PLAT Group.

Patients with peripheral arterial disease have a high risk of death from cardiovascular events. As defective fibrinolysis associated with leg atherosclerosis has been suggested as a predisposing factor, we sought a relation among decreased fibrinolysis, the presence of leg atherosclerosis and the incidence of thrombotic events in a case-control study nested in the PLAT. Fifty-eight patients with coronary and/or cerebral atherothrombotic disease, free of leg atherosclerosis at Doppler examination, were compared with 50 atherosclerotic patients with leg involvement. High D-dimer (153.0 vs 81.3 ng/ml, p < 0.001) and tPA antigen before venous stasis (14.4 vs 11.8 ng/ml, p < 0.03), and low tPA antigen (6.7 vs 15.6 ng/ml, p < 0.01) and fibrinolytic activity released after venous stasis (fibrinolytic capacity: 113.2 vs 281.4 mm2, p < 0.001) were found in patients with leg atherosclerosis. D-dimer and fibrinolytic capacity, in addition to age, were selected by stepwise discriminant analysis as characterizing patients with leg atherosclerosis. Moreover, higher D-dimer and tPA inhibitor characterized patients with leg atherosclerosis who subsequently experienced thrombotic events. These findings constitute evidence of high fibrin turnover and impaired fibrinolytic potential in patients with leg atherosclerosis. Thus impaired fibrinolysis may contribute to the prothrombotic state in these patients.

Stage dependent enhanced induction of hepatocellular carcinomas in rats administered a second dose of diethylnitrosamine.

An experiment was performed to investigate whether the incidence of chemical carcinogen-induced liver tumors in rats is increased by administration, during promotion, of a second dose of carcinogen. Seven groups of male Fischer-344 rats were administered a dose of diethylnitrosamine (DEN), and were placed, 1 month thereafter, on a liver-tumor promotion-regimen, consisting of a choline-devoid diet containing 0.06% phenobarbital. A second dose of the carcinogen was administered, at monthly intervals of promotion, to some of the groups of animals. The experiment was terminated after 7 months of promotion, and the incidence of hepatic tumors was determined histopathologically. In rats administered the second dose of carcinogen after 2 months of promotion, the incidence of hepatocellular carcinomas was 2-3 times greater than that in the other groups of rats. The results are consistent with the proposal, advanced by others, that development of hepatocellular carcinomas may be a multi-hit process, and indicate that, after the first induced at initiation, further genomic alteration(s) may have to occur at very discrete stage(s) in the evolution of initiated cells to tumors.

Effects of dietary choline deficiency on the mutagenic activation of chemical carcinogens by rat liver fractions.

Salmonella mutagenesis assays were used to evaluate the mutagenicity of several chemical carcinogens as mediated by liver S-9 fractions from rats fed a choline-supplemented (CS) or choline-devoid (CD) diet. The liver S-9 fraction from CD diet-fed rats was found to have a significantly decreased ability to activate 2-acetylaminofluorene (2-AAF), 2-aminoanthracene (2-AA) and 6-aminochrysene (6-AC), but not N-hydroxy-2-acetylaminofluorene (HO-N-2-AAF) and dimethylnitrosamine (DMN). The same liver S-9 fraction was also less effective in deactivating N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) but not methylnitrosourea (MNU). A decrease (20%) in the cytochrome P-450 content was found in liver microsomes of CD diet-fed rats. Although it has been shown that feeding a CD diet to rats enhances chemical hepatocarcinogenesis, the data presented here suggest that CD diet does not increase the activation of the chemical procarcinogens tested.

Induction of foci of altered hepatocytes by a single injection of azaserine to rats.

The induction of foci of altered, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes by azaserine was investigated. After injection of a single dose of azaserine, many foci developed in male Wistar rats fed a choline-devoid (CD) diet containing acetylaminofluorene (AAF), but only a few in rats fed a choline-supplemented (CS) diet containing AAF. Similar results were obtained in rats fed a plain CD diet or a plain CS diet and injected with a single dose of azaserine after a partial hepatectomy. These findings indicate that azaserine is an effective initiator of liver carcinogenesis in rats, and that a CD diet acts as a strong promoter of the evolution initiated liver cells to foci of altered, GGT-positive hepatocytes.

Effects of varying the fat content of a choline-devoid diet on promotion of the emergence of gamma-glutamyl-transpeptidase positive foci in the liver carcinogen-treated rats.

Previous studies have shown that feeding a choline-devoid (CD) diet to rats is an effective promoter of liver carcinogenesis. In the present studies, we investigated the effects of CD diets containing different levels of fat (high, 15%; low, 4%) on the induction of foci of gamma-glutamyltranspeptidase (GGT) positive hepatocytes in the liver of rats initiated with a single dose of diethylnitrosamine. In rats fed the high-fat CD diet for 4--6 weeks, a greater number of foci was induced than in rats fed similarly the low-fat CD diet. However, liver DNA synthesis and the rate of hepatocyte mitosis were not significantly different in rats fed the 2 CD diets, but were significantly higher than in rats fed a choline-supplemented diet. These results indicate that beside stimulation of liver cell proliferation, other factor(s) determine the efficacy with which a CD diet exerts its promoting action.

Modulation of tumor promotion in liver carcinogenesis.

Earlier, we demonstrated that feeding to rats a diet devoid of choline, a lipotropic factor, markedly enhances hepatoma induction by several chemical carcinogens, and that the diet acts as a strong promoter of the evolution of initiated cells to foci of altered hepatocytes. The ability of several factors to modulate the action of the choline-devoid (CD) diet as a promoter was investigated by quantitating the foci of gamma-glutamyl transpeptidase-positive hepatocytes developed in rats exposed to a single injection of diethylnitrosamine. Addition to the diet of phenobarbital (PHB) resulted in a promoting action stronger than those of the CD diet or of PHB alone. Two other barbiturates, amobarbital (AMB) and pentobarbital (PTB) exerted an effect similar to that of PHB, while barbituric acid (BA) had no effect. In other studies, lowering the fat content of the CD diet reduced its efficacy as a promotor, while the addition of a hypolipidemic agent, BR931, 4-chloro-6-(2,3 xylidino)-2-pyrimidinylthio (N-beta-hydroxy-ethyl)acetamide, completely abolished the promoting action of the CD diet. In rats not exposed to carcinogen, feeding the CD diet caused a marked enhancement of liver DNA synthesis and of cell proliferation. Inclusion of PHB, PTB or AMB in the CD diet inhibited these effects, while BA exerted no inhibition. The increased rate of DNA synthesis and cell proliferation in the liver were not affected by the level of fat in the CD diet. These results suggest that beside a stimulation of liver cell proliferation, other factor(s) determine the efficacy with which a CD diet exerts its promoting action.

Conditions under which chlordiazepoxide influences gustatory contrast.

Rats shifted from 32% sucrose to 4% sucrose consumed less 4% than animals without prior experience with 32% sucrose. The influence of chlordiazepoxide (CDP) on this successive negative contrast obtained in sucrose ingestion was investigated in four experiments. The results indicated that (1) rats injected with CDP during both preshift experience with 32% sucrose and post-shift experience with 4% sucrose showed an essentially unchanged contrast effect compared with saline-injected rats, (2) CDP injection for the first time on post-shift day 2 eliminated contrast but post-shift day 1 injections had little effect, (3) animals injected with CDP throughout preshift and switched to saline coincident with the sucrose shift showed a contrast effect at least as great as control animals, and (4) injections of CDP tended to elevate lick rate regardless of other conditions. These results indicate a disinhibitory effect of CDP and possible neophobia operating on the first post-shift day.

Diffuse arachnoiditis following epidural analgesia.

Four cases of spinal arachnoiditis are reported, which occurred as a delayed complication of epidural anaesthesia. Different causes are considered: the most convincing hypothesis is that there was a subarachnoid hyperergic reaction to the drugs injected during epidural anaesthesia.