TGF-ß/YB-1/Atg7 axis promotes the proliferation of hepatic progenitor cells and liver fibrogenesis.

Hepatic fibrosis is characterized by excessive extracellular matrix deposition and ductular reactions, manifested as the expansion of hepatic progenitor cells (HPCs). We previously reported that the Y-box binding protein 1 (YB-1) in HPCs is involved in chronic liver injury. In this study, we constructed YB-1f/f Foxl1-Cre mice and investigated the role of YB-1 in HPC expansion in murine choline-deficient, ethionine-supplemented (CDE), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) models. Liver injury and fibrosis were measured using hematoxylin and eosin (HE), Masson, and Sirius Red staining. HPC proliferation was detected using EdU and immunofluorescence (IF). Autophagic flow was measured by mCherry-GFP-LC3B staining and transmission electron microscopy (TEM). YB-1 expression was measured by immunofluorescence and western blotting. CUT & Tag analysis, chromatin immunoprecipitation, and RT-PCR were performed to explore the regulation of autophagy-related protein 7 (Atg7) transcription by YB-1. Our results indicated that liver injury was accompanied by high expression of YB-1, proliferative HPCs, and activated autophagy in the CDE and DDC models. YB-1f/f Cre+/- mice displayed less liver injury and fibrosis than YB-1f/f Cre-/- mice in the CDE and DDC models. YB-1 promoted proliferation and autophagy of HPCs in vitro and in vivo. Transforming growth factor-ß (TGF-ß) induced YB-1 nuclear translocation and facilitated the proliferation and autophagy of HPCs. YB-1 nuclear translocation promoted the transcription of Atg7, which is essential for TGF-ß/YB-1 mediated HPCs expansion in vitro and in vivo. In summary, YB-1 nuclear translocation induced by TGF-ß in HPCs promotes the proliferation and autophagy of HPCs and Atg7 participates in YB-1-mediated HPC-expansion and liver fibrosis.

Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.

UNLABELLED: In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area, migrate inside the lobule, and undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. We analyzed LPC expansion and matrix accumulation in a choline-deficient, ethionine-supplemented (CDE) model of LPC proliferation. After day 3, CDE induced collagen deposits in the periportal area. Expansion of LPCs as assessed by increased number of cytokeratin 19 (CK19)-positive cells was first observed at day 7, while ECM accumulated 10 times more than in controls. Thereafter, LPCs and ECM increased in parallel. Furthermore, ECM not only accumulates prior to the increase in number of LPCs, but is also found in front of LPCs along the porto-venous gradient of lobular invasion. Double immunostaining revealed that LPCs are embedded in ECM at all times. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by alpha-smooth muscle actin (alpha-SMA)-positive cells. Gene expression analyses confirmed these observations. The expression of CK19, alpha-fetoprotein, E-cadherin, and CD49f messenger RNA (mRNA), largely overexpressed by LPCs, significantly increased between day 7 and day 10. By contrast, at day 3 there was a rapid burst in the expression of components of the ECM, collagen I and laminin, as well as in alpha-SMA and connective tissue growth factor expression. CONCLUSION: Our data demonstrate that, in a CDE model, ECM deposition and activation of matrix-producing cells occurred as an initial phase, prior to LPC expansion, and in front of LPCs along the porto-venous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation and differentiation of liver progenitor cells.

Analysis of time-related metabolic fluctuations induced by ethionine in the rat.

The time-course of metabolic events following response to a model hepatotoxin ethionine (800 mg/kg) was investigated over a 7 day period in rats using high-resolution (1)H NMR spectroscopic analysis of urine and multivariate statistics. Complementary information was obtained by multivariate analysis of (1)H MAS NMR spectra of intact liver and by conventional histopathology and clinical chemistry of blood plasma. (1)H MAS NMR spectra of liver showed toxin-induced lipidosis 24 h postdose consistent with the steatosis observed by histopathology, while hypertaurinuria was suggestive of liver injury. Early biochemical changes in urine included elevation of guanidinoacetate, suggesting impaired methylation reactions. Urinary increases in 5-oxoproline and glycine suggested disruption of the gamma-glutamyl cycle. Signs of ATP depletion together with impairment of the energy metabolism were given from the decreased levels in tricarboxylic acid cycle intermediates, the appearance of ketone bodies in urine, the depletion of hepatic glucose and glycogen, and also hypoglycemia. The observed increase in nicotinuric acid in urine could be an indication of an increase in NAD catabolism, a possible consequence of ATP depletion. Effects on the gut microbiota were suggested by the observed urinary reductions in the microbial metabolites 3-/4-hydroxyphenyl propionic acid, dimethylamine, and tryptamine. At later stages of toxicity, there was evidence of kidney damage, as indicated by the tubular damage observed by histopathology, supported by increased urinary excretion of lactic acid, amino acids, and glucose. These studies have given new insights into mechanisms of ethionine-induced toxicity and show the value of multisystem level data integration in the understanding of experimental models of toxicity or disease.

In vivo antihepatotoxic effects of Ligularia fischeri var. spiciformis and the identification of the active component, 3,4-dicaffeoylquinic acid.

Pretreatment with a methanolic extract of Ligularia fischeri var. spiciformis (Compositae) herb inhibited hepatotoxicities caused by CCl4, D-galactosamine (GalN), alpha-naphthylisothiocyanate (ANIT), and DL-ethionine in rats. An ethyl acetate (EtOAc) extract fractionated from the methanolic extract showed a strong inhibitory effect. A major component, 3,4-dicaffeoylquinic acid (DCQA), isolated from the methanolic extract was examined for antihepatotoxicity. Pretreatment with DCQA (5 and 10 mg/kg, p.o.) significantly reduced serum aminotransferases (alanine and aspartate), sorbitol dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, and lactate dehydrogenase activities during CCl4- or GalN-induced hepatotoxicity, suggesting that DCQA is a major principle for the antihepatotoxic activity of L. fischeri var. spiciformis. DCQA also partially restored bile flow and reduced total bilirubin and cholic acid concentrations in rats with ANIT-induced cholestasis. Treatment with DCQA inhibited the increase in triglyceride, cholesterol, and total lipids in DL-ethionine-induced fatty liver. These results support the traditionally held belief that this plant can be used for the treatment of jaundice and hepatic failure.

Natural disruption of group 2 phospholipase A2 gene protects against choline-deficient ethionine-supplemented diet-induced acute pancreatitis and lung injury.

OBJECTIVES: Group 2 phospholipase A2 (PLA2) plays an important role in the pathogenesis of multiple organ failure associated with acute pancreatitis. C57 BL/6J mice are natural group 2 PLA2 knockout mice lacking group 2 PLA2 mRNA. To clarify the role of group 2 PLA2 in the exacerbation of acute pancreatitis, we studied the biologic and histologic alterations in choline-deficient and ethionine-supplemented (CDE) diet-induced pancreatitis in group 2 PLA2-deficient C57 BL/6J mice and compared them with those in wild-type mice. METHODS: Female C57 BL/6J mice weighing 20 to 22 g were fed a CDE diet for 3 days to induce pancreatitis. Female C3H/HEJ mice were used as controls. Mice were killed on days 1, 2, and 3 after the onset of the CDE diet. The severity of pancreatitis was evaluated by survival rate, plasma PLA2 activity, serum amylase level, histologic changes in the pancreas and lung, and myeloperoxidase activity in the lung. RESULTS: The survival rate of C57 BL/6J mice was 100% up to day 3 after the onset of the CDE diet, whereas that of the control mice was 42% on day 3. Plasma PLA2 activity in control mice increased on day 3 but did not increase in C57 BL/6J mice. Serum amylase activity on day 3 in C57 BL/6J mice was 15,480 +/- 3036 SU/dL, which was significantly lower than that in the control mice (43,760 +/- 8657 SU/dL, P < 0.01). Histologic changes in the pancreas of C57 BL/6J mice were markedly milder than in control mice. The degree of alveolar membrane thickening and infiltration of inflammatory cells in the lung of C57 BL/6J mice were overtly less than those of the controls. Myeloperoxidase activity in the lung of C57 BL/6J mice was lower, albeit insignificant, than in C3H/HEJ mice. CONCLUSIONS: Natural disruption of the group 2 PLA2 gene protects against CDE diet-induced acute pancreatitis and associated lung injury. These findings support the view that group 2 PLA2 is one of the factors in the exacerbation of severe acute pancreatitis.

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member gamma, but not alpha or delta.

Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.

Protective effect of soybean against hepatocarcinogenesis induced by DL-ethionine.

There has been increasing interest in the value of using soybean to delay or reduce the tumor incidence. This study was undertaken to investigate the possible protective effects of soybean against hepatocarcinogenesis induced by DL-ethionine. Accordingly, we measured biochemical changes occurring in serum and liver of rats treated with DL-ethionine in the presence or absence of soybean. Male albino rats were fed a control diet containing the hepatocarcinogen, DL-ethionine, or the control diet plus soybean 30%, or the control diet plus soybean plus DL-ethionine 0.25% for three months and then returned to a control diet for up to nine months. Rats fed a control diet plus DL-ethionine showed a gradual decrease in liver DNA, RNA, total protein, and liver weight and enzyme activities of liver transaminases (GOT and GPT) and alkaline phosphatase over the 7-month study period. This was followed by a large increase in the liver parameters at the end of the 9(th) month, except for 5'-nucleotidase and glucose-6-phosphatase that showed a large decrease. On the other hand, a gradual increase in the serum enzyme activities of GOT, GPT, 5-nucleotidase, alkaline phosphatase, and in the albumin/globulin (A/G) ratio is observed in the group of rats fed a control diet plus DL-ethionine compared to the control group over 8 months, and this was followed by a large increase in all serum parameters studied at nine-months. The administration of 30% soybean to the rat diet in addition to DL-ethionine maintained all parameters studied at near control values until the end of the 9(th) month. This study suggests that soybean has a protective effect against the hepatocarcinogenesis induced by DL-ethionine.

Inhibitory effects of N,N'-diphenyl-p-phenylenediamine on the early stage of the enhanced hepatocarcinogenesis caused by coadministration of ethionine and a choline-deficient L-amino acid-defined diet in rats.

Effects of N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, on liver carcinogenesis caused by a choline-deficient L-amino acid-defined (CDAA) diet containing ethionine were studied in Fischer 344 rats. Male animals, 6 weeks old, were fed a CDAA diet, a choline-supplemented L-amino acid-defined (CSAA) diet or a CDAA diet containing 0.05% ethionine with or without 0.2% DPPD. Histological changes and lesions positive for gamma-glutamyltransferase (GGT) were analyzed 12 weeks after the beginning of the experiment. The levels of 8-hydroxyguanine (8-OHGua) in DNA and 2-thiobarbituric acid-reacting substances (TBARS) were measured as the parameters for cellular oxidative damage after 4 and 11 days of treatment. Expression of c-myc and c-Ha-ras was also investigated in relation to cell proliferation after 2, 4, 8 and 11 days. Histologically, development of diffuse fatty liver observed in rats fed a CDAA diet was inhibited, while massive oval cell proliferation and cholangiofibrosis resulted from the addition of ethionine with/without DPPD. The sizes but not numbers of GGT-positive lesions seen in the liver of rats fed a CDAA diet were increased and the levels of 8-OHGua formation and TBARS generation were also increased by the ethionine supplement. Both numbers and sizes of GGT-positive lesions were decreased and the level of TBARS, but not 8-OHGua, was decreased by adding DPPD. The increased expression of c-myc and c-Ha-ras detected in the liver of rats fed a CDAA diet was further increased by addition of ethionine and again reduced by DPPD. These results indicate that an antioxidant DPPD can inhibit the early stage of enhanced hepatocarcinogenesis caused by coadministration of ethionine and a CDAA diet, by blocking cellular oxidative damage as well as c-myc and c-Ha-ras expression.

Differentiation of oval cells into duct-like cells in preneoplastic liver of rats placed on a choline-deficient diet supplemented with ethionine.

Feeding male Wistar rats a choline-deficient diet containing 0.07% DL-ethionine (CDE diet) for up to 5 weeks results in the production of two distinct non-parenchymal cell populations, oval and duct-like cells. These cells can undergo replication and display different patterns of expression of glutathione S-transferases (GSTs) and pyruvate kinases (PKs). Oval cells were first detected around the periportal region after 1 week of CDE treatment and infiltrated the parenchyma after 2 weeks. Duct-like structures first appeared as isolated ducts in the parenchymal region at 2 weeks and were easily detected after 2.5 weeks. These duct-like structures differed from the bile ducts which reside in the portal region. Large concentrations of duct-like structures in cyst-like clusters were detected after 5 weeks. Enlargement of these structures from single ducts to clusters of up to 20 ducts was observed over 3-5 weeks of CDE treatment. The number of cells forming a duct increased from 5 to 30 cells. We established a double immunocytochemical staining technique to characterize the oval and duct-like cells for their expression of GSTs and PKs. pi GST and M2-PK, which are fetal hepatocytes isoenzymes, are present in virtually all the oval and duct-like cells. Most of the oval cells are devoid of the adult hepatocytes markers, alpha GST, mu GST and L-PK. There are two sub-populations of duct-like cells, one which expresses only fetal markers and the other which co-expresses the adult and fetal isoenzymes. Hence, oval cells display characteristics of fetal hepatocytes and some duct-like cells appear more mature than oval cells. Using a combination of double immunocytochemical and [3H]thymidine labelling techniques we have established that oval cells differentiate into duct-like cells.

Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.

Liver phosphatidylcholine hydroperoxidation provoked by ethionine-containing choline-deficient diet in mice.

It is shown that peroxidation of phosphatidylcholine (PC) is enhanced in liver of mice fed a hepatocarcinogenic choline-deficient diet containing 0.1% w/w ethionine. Mice were divided into 4 groups and fed for 4 weeks one of the following diets: choline-supplemented; choline-supplemented containing ethionine; choline-deficient; and choline-deficient containing ethionine. Phosphatidylcholine hydroperoxide (PCOOH) of liver lipids was measured by high performance liquid chromatography using a chemiluminescence detector. Mice fed a choline-deficient diet containing ethionine showed 6-fold higher PCOOH levels than the choline-supplemented control mice: the PCOOH/PC molar ratios of liver lipids were 32.3 X 10(-5) and 5.6 X 10(-5), respectively. In addition to this remarkable degree of lipid peroxidation in liver of mice fed the choline-deficient diet containing ethionine, we also observed a significant liver fatty infiltration, a decrease in plasma and liver alpha-tocopherol, and an increase in liver injury-indicative enzyme activities. Also, marker enzymes for hepatocarcinogenesis, glucose-6-phosphatase and gamma-glutamyl transpeptidase were affected. These data suggest that enhanced hydroperoxidation of phosphatidylcholine may participate in hepatocarcinogenesis provoked by choline deficiency in the presence of ethionine.

[The effect of pollen on the changes in the liver of laboratory rats evoked by ethionine, carbon tetrachloride, allyl alcohol and galactosamine]. / Der Einfluss von Pollen auf die von Ethionin, Tetrachlorkohlenstoff, Allylalkohol und Galactosamin experimentell hervorgerufenene Veränderungen in der Leber von Laboratoriumsratten.

Doses of 50 mg/kg body weight and 200 mg/kg of Cernitin T 60 and Cernitin GBX may be used over 14 days for effective protection of rat liver cells from toxic action of ethionine. Application of CCl4 caused damage to the liver of rats. Such damage may be mitigated by both Cernitin preparations, particularly by Cernitin T 60. The damage was further reduced by Cernitin, following administration of allyl alcohol, with increase in transaminase, phosphatase, and bilirubin activities being used as criteria for measurement. The liver-protecting effect of Cernitin was confirmed in histopathological investigations. Cernitins prevented much of the damage actually caused by galactosamine.

Amelioration of ethionine toxicity in the chick.

Several chick bioassays were conducted to evaluate means of ameliorating ethionine toxicity. Supplementing a corn-soy diet marginally deficient in sulfur amino acids (methionine + cystine) with .075% D,L-ethionine reduced weight gain in 8-day-old chicks by 70% compared to gains of unsupplemented controls. Dietary addition of .50% DL-methionine prevented reduction in weight gain and feed intake resulting from ethionine supplementation whereas feeding supplemental L-cystine was without effect. Supplementation of the ethionine-containing diet with either choline or betaine ameliorated the growth depression, although neither compound was able to completely overcome the toxic effects of ethionine. Dietary ethionine did not affect plasma levels of free methionine or cystine but did increase plasma free glycine 6-fold. Dietary addition of .50% DL-methionine caused normalization of plasma glycine levels whereas it elevated plasma methionine concentration. Although results suggested the possibility of ethionine-induced serine or threonine deficiency, dietary additions of .75% L-serine or .75% L-threonine failed to improve chick weight gain. These studies suggest that ethionine, in addition to affecting transsulfuration and transmethylation activity may exert specific effects on certain amino acids in tissue pools.

Ethionine carcinogenesis in CD-1, BALB/c and C3H mice.

In two separate in vivo studies, ethionine was evaluated for carcinogenic activity in mice. In the first study, DL-ethionine was fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and 0.25% (high dose, HD) concentrations to the following groups of mice (30 animals/group): Swiss Webster CD-1 females, BALB/c males, and C3H/HeN males and females. Because of severe toxicity, BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine. The Swiss and BALB/c mice were maintained on their respective diets for up to 105 weeks before killing whereas the C3H mice were killed at 68 weeks because of the high spontaneous incidence of liver tumors in this strain. The percentages of animals at risk (surviving the time to the first liver tumor recorded in each sex and strain) that bore liver tumors were as follows: Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23); Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23); C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H female controls, 5% (1/20); C3H female LD, 60% (12/20); C3H female HD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c male LD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls, 0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c female HD, 92% (12/13). The female mice were more responsive than the males in developing liver tumors. The results of the feeding study are compared with those obtained in a second study in which C3H female mice were intubated with 0, 150 or 500 mg DL-ethionine/kg body wt three times per week for 30 weeks and killed at 2 years. Only the LD mice showed a significantly increased incidence of liver tumors (20/39) as compared to controls (12/41) or HD mice (7/37) in the latter study. The hepatic levels of the major ethionine metabolite and methylase inhibitor, S-adenosylethionine (AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine (AdoMet) were determined in Swiss female mice fed either 0.1 or 0.3% in the diet for 1-6 weeks. Hepatic AdoEt levels ranged from 37 to 80 micrograms/g liver in the LD animals and from 61 to 203 micrograms/g liver in the HD group; levels of the endogenous metabolite AdoMet correspondingly dropped to 65% of the normal levels. The present results (i) extend to different strains and to both sexes previous observations demonstrating the hepatocarcinogenic activity of ethionine in mice; and (ii) indicate that as in the rat such activity may be exerted through the formation of AdoEt.

Survey of various chemicals for initiating and promoting activities in a short-term in vivo system based on generation of hyperplastic liver nodules in rats.

The effects of pre- (initiation stage) and post- (promotion stage) administration of 17 chemical carcinogens (including both hepatic and non-hepatic carcinogens), 3 promoters and 2 non-carcinogenic analogs, on the induction of liver hyperplastic nodules were investigated in rats. Test chemicals were administered during the initiation stage after which rats were fed dietary N-2-fluorenylacetamide (2-FAA) for 2 weeks in conjugation with necrogenic CCl4 to enhance production of nodules initiated by test chemicals. Alternatively, effects of test chemicals administered during the promotion phase were evaluated in rats given a combination of dietary 2-FAA for 2 weeks and necrogenic CCl4. This initiation regimen resulted in very few nodules unless a promoter was subsequently used. All chemical carcinogens administered during the initiation stage induced more frequent, larger hyperplastic nodules than did control treatments. However, neither the promoters nor the non-carcinogenic analogs induced hyperplastic nodules if they were administered during the initiation stage. In contrast, hepatocarcinogens and a promoter of hepatocarcinogenesis (phenobarbital) administered in the promotion stage had enhancing (promoting) activity on hyperplastic liver nodules, whereas non-hepatocarcinogens, a promoter for skin carcinogenesis (12-O-tetradecanoylphorbol-13-acetate) and a promoter for urinary bladder carcinogenesis (saccharin) did not. Non-carcinogenic analogs were not active when administered during either the initiation or promotion stages. These findings demonstrated the utility of employing short-term in vivo assays for both initiating and enhancing (promoting) activities of chemicals. By our system, chemicals were classified into 4 main groups, namely, (i) hepatocarcinogens which have both initiating and enhancing activity, (ii) non-hepatocarcinogens with initiating activity only, (iii) promoters of hepatocarcinogenesis which have promoting activity and (iv) non-carcinogens and non liver promoters which did not initiate nor promote nodule development. This is a more discriminating means of assaying carcinogenic activity than is possible with short-term in vitro screening assays for mutagenicity.

The influence of DL-methionine on the metabolism of S-adenosylethionine in rats chronically treated with DL-ethionine.

The concentration of S-adenosylethionine in the liver of ethionine-fed rats was increased gradually during the process of carcinogenesis. This increase may have been due to the decreased capacity of the treated rats to acetylate ethionine sulfoxide. Ethionine sulfoxide is considered as the main reserve pool of ethionine for the synthesis of S-adenosylethionine. When the ethionine diet was supplemented by DL-methionine (0.3 to 0.9%), the increase in the concentration of S-adenosylethionine during the period of observation (28 to 150 days) was lower and the acetylation of ethionine sulfoxide was significantly higher. The concentration of the total S-adenosyl compounds in the liver of rats on a diet supplemented with DL-methionine was increased over the concentration of S-adenosylethionine in rats fed ethionine alone, and the S-adenosylethionine portion of this fraction was only about 30% lower. The supplementation of the diet with methionine restored the diurnal oscillation of adenosine 5'-triphosphate in the liver, which had been absent in rats ingesting only ethionine.