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Hepatic cell injury was studied in an in vitro system using rat liver slices incubated in two stages. During the first 2 hr slices were exposed to 10 mM paracetamol, this was absent during the subsequent 4 hr of incubation. Cell damage was quantified at the end by measuring leakage of lactic dehydrogenase, increase in water content and potassium loss. Treatment of slices with 20 mM fructose in the second period of incubation prevented paracetamol-induced damage. The effect of fructose was not modified by the continued presence of paracetamol in the second incubation period. The inhibition of glycolysis either with 1 mM NaF or 10 microM iodoacetate blocked the effect of fructose. The protective effect afforded by fructose was not duplicated by the addition of lactate. All these findings strongly suggest an increase in intracellular ATP levels as the most probable explanation for the protective effect of fructose, and point to fructose as a potentially useful therapeutic tool for protection of the liver late in paracetamol intoxication.

Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and alpha-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.

To determine changes in parathyroid hormone secretion and target organ response caused by acute pancreatitis before the development of systemic toxic conditions, experimental acute pancreatitis was induced in rats with a choline-deficient, ethionine-supplemented diet. After 7 days, the rats were weighed and bled, and one kidney was assayed for 25-hydroxyvitamin D1 hydroxylase activity. Several manifestations of pancreatitis were observed in rats given the diet: weight loss (from 29.6 to 26.3 g vs that for control rats, from 29 to 52.8 g) and lower dietary intake (15.5 vs 47 g per rat per 7 days). Serum amylase levels fell from 1794 to 350 U/L in rats given the choline-deficient, ethionine-supplemented diet compared with levels of 1800 to 2100 U/L in control rats. The pancreases of rats given the choline-deficient, ethionine-supplemented diet showed degeneration, necrosis, and hemorrhaging. Serum levels of calcium, phosphorus, chloride, and parathyroid hormone did not change significantly throughout the experiment. Renal 25-hydroxyvitamin D1 hydroxylase activity was higher than in control rats (8.9 +/- 0.8 vs 7.6 +/- 0.6 fmol/mg of kidney per minute). Acute pancreatitis in this experimental animal model does not alter serum levels of calcium and parathyroid hormone or reduce target organ responsiveness to the hormone.

Human gingival fibroblast cultures were used to investigate the role of cellular thiol redox status in the mitogenic response. Increases in intracellular Ca2+ and cell cycle progression beyond G1 were followed as parameters of cellular mitogen-induced responses. Ethionine provided a G1 stage synchronization and altered the cellular redox poise as measured by the ratio NAD(P)H/NAD(P)+. Cultures harvested immediately after the 6 day ethionine low-serum synchronization showed a significant oxidation of their redox poise. Synchronized cultures, which were also glutathione (GSH) depleted, still showed an oxidized redox poise and significantly reduced GSH levels following a 24 hr incubation in drug-free, rich medium. Cellular reduced nicotinamide nucleotide levels correlated strongly (r = 0.995) with capacity to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). The sustained mitogenic response, as determined by cell cycle progression beyond G1, was also found to be interrelated with the cellular thiol redox status. Following a 24 hr recovery incubation in serum-rich medium, formerly synchronized cultures showed a rebound of their redox poise to a more reduced state and significant cell cycle progression beyond G1. In contrast, synchronized, GSH-depleted cultures did not progress and showed population distributions similar to those of cultures harvested immediately postsynchronization. Upon recovery of cellular GSH and reduced nicotinamide nucleotide levels, formerly GSH-depleted, growth-arrested cultures resumed cell cycle progression. The results suggest that the cellular response to specific mitogens is interrelated with the cellular thiol redox status. Cells that possess a thiol redox status below a threshold response point may have compromised Ca2+ sequestration and/or mobilization and therefore may be incapable of initiating the mitogen induced response cascade that culminates in cell cycle progression.

1. The morphological changes of the pancreas induced by administration of dl-ethionine were determined for treated rats and for a group of pair-fed untreated controls in order to separate the direct effect of dl-ethionine from the effect of accompanying reduction of food intake. 2. Adult male Wistar rats were studied in 3 groups of 10 animals each: 1) fed ad libitum and treated daily with dl-ethionine (35 mg/100 g body weight, ip) for 10 days; 2) treated daily with vehicle (saline) as group 1 and pair-fed to group 1; 3) treated daily with vehicle (saline) and fed ad libitum. Two rats from each group were killed on days 2, 4, 6, 8 and 10 to characterize the pancreas in terms of morphological properties using morphometric analysis. 3. Exposure to dl-ethionine induced a progressive and significant decrease in both pancreas weight and acinar cell number and volume. Pair feeding induced less pronounced decreases in pancreas weight and acinar cell volume. Pancreas weight was 125 mg/100 g body weight for dl-ethionine-treated vs 205 mg/100 g body weight for pair-fed controls and 320 mg/100 g body weight for ad libitum-fed controls after 10 days. Acinar cell number (x +/- sigma/square root of n): 175 x 10(6) +/- 28.4 per micron3 for dl-ethionine-treated vs 221 x 10(6) +/- 17.3 per micron3 for pair-fed controls and 271 x 10(6) +/- 23.9 per micron3 for ad libitum-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The exposure of K-562 cells to ethidium bromide or acridine orange as DNA-intercalating agents induced their differentiation to hemoglobin-producing cells. The addition of L-ethionine or dimethylsulfoxide induced neither such differentiation nor changes in poly(A) polymerase activity of K-562 cells, though the addition of ethidium bromide, acridine orange, hydroxyurea, or 1-beta-arabinofuranosylcytosine induced erythroid differentiation and decreases in poly(A) polymerase activity. These results suggest that DNA-intercalation is one of the pathways triggering differentiation of K-562 cells and the decrease in poly(A) polymerase activity may be an important change associated with differentiation of K-562 cells.

The effects of FUT-187 (6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoate dimethanesulfonate, CAS 103926-82-5), a novel synthetic protease inhibitor, were examined in experimental rat and canine models of pancreatitis. 1. FUT-187 significantly increased the survival of rats with trypsin- and phospholipase A2-induced pancreatitis in a dose-dependent manner (10-100 mg/kg, p.o.). 2. FUT-187 decreased plasma enzymatic activity reflecting the degree of pancreatitis in rats with ethionine-induced pancreatitis, and showed a tendency to ameliorate histopathological changes in the pancreas (10-100 mg/kg p.o.). 3. FUT-187 (10 mg/kg) produced an obvious improvement of various biochemical parameters of pancreatitis and also reduced histopathological changes in the pancreas in animals with experimental pancreatitis produced by the closed duodenal loop method. In addition, FUT-187 significantly increased the survival of dogs when given by direct administration into the lumen of the closed duodenal loop. The therapeutic effects of FUT-187 in experimental pancreatitis were nearly equal in most instances to those of camostat mesilate. Thus, FUT-187 would appear to be an effective new agent for the treatment of pancreatitis.

A 100% yield of pancreatic hepatocytes was induced in pancreas tissues of female hamsters treated with twice-repeated sequential administrations of DL-ethionine (ethionine) together with a protein-free diet and then L-methionine (methionine) for 10 weeks. The cells were also found in 40% of hamsters receiving 20 mg/kg body weight of the pancreatic carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP) given twice at the peak of pancreatic regeneration stimulated by methionine after ethionine-induced cell damage. However, BOP at doses of 30, 70, and 100 mg/kg body weight administered before the occurrence of pancreatic regeneration dose-dependently inhibited their appearance, with reduction of the yield to 40%, 25%, and 8.3% respectively, and BOP per se did not induce any development of pancreatic hepatocytes. Stein iodine staining revealed bile pigments in the induced hamster pancreatic eosinophilic cell populations.

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic microsomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [35S]Met-tRNAi demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.

The nonhydrolyzable guanyl nucleotide GTP gamma S stimulated phosphoinositidase C activity in two preparations obtained from mouse pancreatic acini labeled with myo[2-3H]inositol: a cell-free membrane fraction and intact electropermeabilized acini. This action was dose-dependent, was shared by other nonhydrolyzable guanyl nucleotides such as GMP-phencyclidine hydrochloride and GMP-PMP, as well as by fluoride, and was calcium-independent. Contrarily, no effect was observed even at doses of GTP gamma S as high as 10 microM when the same protocol was repeated on identical acinar preparations from mice fed a choline-deficient, ethionine-supplemented diet. This regimen is known to uncouple secretagogue-receptor occupancy from inositol 1,4,5-trisphosphate generation in pancreatic acinar cells and lead to necrotizing hemorrhagic pancreatitis. These data lead us to conclude that the ethionine-induced inactivation of guanyl nucleotide-dependent pancreatic phosphoinositidase C in pancreatic acinar cells is not the result of either a decrease in GTP level or a decrease in GTP availability. These findings further confirm previous work from this laboratory, which has shown that the biochemical lesion induced by this diet occurs after the agonist-receptor binding step. The diet-induced lesion could be either at the level of the G-protein that couples the enzyme with the receptor or at the level of the phospholipase itself.

To clarify the energy-dependency of biliary excretion of a diagnostic dye, the effect of ATP depletion on biliary excretion was investigated. Biliary excretion of indocyanine green (ICG) in rat liver reached a maximum 20 min after intravenous application. The level of ATP in the liver was lowered to 35.4% of the control value about 5 h after administration of DL-ethionine (100 mg/100 g body weight). The rate of bile flow and the amount of ICG excreted in initial 10 min into the bile of ethionine-treated rats were lowered to 67.5% and 61.3% of the control values, respectively. The amount of ICG excreted for 10 min from 30 min to 40 min after application of ICG in ethionine-treated rats was comparable to that of the control. The level of ICG in blood 5 min after application of ICG was raised by 59.2% of the control value. These results suggest that ATP depletion in the liver caused by treatment with ethionine suppresses the initial rate of uptake and biliary excretion of ICG.

The rapid production model of pancreatic duct adenocarcinoma established previously in female Syrian hamsters was applied to male hamsters to confirm the efficacy of the "augmentation pressure" procedure and to reveal sex difference in response to this procedure. Hamsters received 70 mg/kg N-nitrosobis (2-oxopropyl) amine (BOP) as an initiation and two cycles of "augmentation pressure" consisting of ethionine on a choline-deficient diet, methionine and BOP and an additional 20 mg/kg BOP and were killed 10 weeks after the beginning of the experiment. During the experimental period, 28% of the hamsters died and severe body weight loss was observed, however, incidence of pancreatic carcinomas reached 50% which is a significantly higher level than that in hamsters which received BOP without "augmentation pressure". A 66.7% yield of cholangiocellular tumors was also observed. The present result indicates that the "augmentation pressure" effected rapid production of pancreatic carcinoma in male hamsters in spite of sex difference in response.

Normal human squamous mucosal cells of the soft palate, buccal surface, epiglottis, hypopharynx, floor of the mouth, and tongue were cultured in vitro in serum-free medium. In medium MCDB 153 containing epidermal growth factor, insulin, bovine pituitary extract, and 0.1 or 2.0 mmol/L Ca++, squamous mucosal cells double every 24 hours. These cells then can be induced to arrest their proliferation reversibly by treatment with transforming growth factor-beta or ethionine, and they can irreversibly growth-arrest during senescence or when cultured in growth factor-deficient medium containing 2 mmol/L Ca++. The latter medium also induces differentiation, as does culture of cells in serum-containing medium. Serum-containing medium furthermore promotes extensive cell stratification and the formation of multilayered squamous mucosal tissue specimens that can be removed intact by Dispase treatment. These specimens represent potential autogenous mucosal grafts that can be used in patients who require reconstructive surgery of the oral cavity and oropharynx. Normal human squamous mucosal cells therefore closely resemble keratinocytes derived from the epidermis in the mechanisms that regulate proliferation and differentiation. This model cell system should facilitate future studies on upper aerodigestive tract squamous mucosal cell physiology and pathophysiology.

The hypothesis that liver carcinogenesis may have as an important facet the early selection of carcinogen-resistant cells was tested in animals in which putative premalignant hepatocyte populations, hyperplastic nodules, were induced by 2-acetylaminofluorene or by ethionine. Hyperplastic nodules were observed to be resistant to the acute necrogenic effects of 2 hepatotoxins, CCl4 and dimethylnitrosamine, under conditions in which liver cell necrosis occurred in the liver surrounding the nodules. In addition, although [methyl-3H]dimethylnitrosamine was taken up to an equal degree in nodules and normal liver, the interactions with DNA, RNA, and protein in hyperplastic nodules were found to be about 50% less than in control liver. Hyperplastic nodules showed a marked decrease in uptake of [9-14C]-2-acetylaminofluorene, a finding that could account for the large decrease in labeling of DNA, RNA, and protein by [9-14C]-2acetylaminofluorene observed in the nodules. The results are consistent with and support the hypothesis that new hepatocyte populations that appear prior to cancer, during liver carcinogenesis, have as an important biological property a resistance to the cytotoxic effect of hepatocarcinogens. The basis for this resistance might be a decrease in uptake and/or a reduction in the level of activation of carcinogens.

Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins.

The influence of various inhibitors of hyphal growth, sporulation and biosynthesis of aflatoxin B1 in Aspergillus parasiticus NRRL2999 was studied. 6-Thioguanine, DL-ethionine, fluoroacetic acid and phenylboric acid, inhibitors of maturation of fungal conidiophores and of conidiogenesis, were added at various concentrations to malt extract agar. Lower concentrations of 6-thioguanine and DL-ethionine did not inhibit the growth of hyphae and the sporulation. Phenylboric acid reduced conidiogenesis more than hyphal growth. The yields of aflatoxin B1 were significantly reduced. Additions of fluoroacetic acid did not greatly affect the growth of hyphae but totally inhibited the production of conidia and concurrently significantly reduced the formation of aflatoxin B1. An interrelation between conidiogenesis and onset of secondary metabolism in A. parasiticus is evident.

The effect of methylation antagonists (DL-ethionine, S-adenosyl homocysteine and folic acid analogs-aminopterine, ametopterine and tomisine) on vitamin B12 and porphyrin biosynthesis of P. shermanii resting cells was studied. DL-ethionine proved the most active inhibitor. Inhibition of vitamin B12 biosynthesis induced by DL-ethionine was accompanied by increase in porphyrin formation. DL-methionine reversed the inhibitory effect of DL-ethionine on the vitamin synthesis. Other compounds tested showed a less marked effect.

Methionine analogs such as ethionine, selenomethionine, and trifluoromethionine all arrest growth and division of the yeast Saccharomyces cerevisiae. One analog, ethionine, caused cells of the yeast to arrest specifically within G1; reciprocal shift experiments showed that ethionine and alpha-factor arrested cells at the same step ("start"). The major effect of ethionine on synthesis of macromolecules was to reduce both the rate of appearance of 35S ribosomal precursor RNA and the rate of production of mature rRNA. Synthesis of protein was relatively unaffected by ethionine. Selenomethionine and trifluoromethionine caused cells to arrest randomly in the cell division cycle. Although treatment of cells with either selenomethionine or trifluoromethionine also reduced the rate of total RNA synthesis, each of these analogs had other effects that presumably prohibited completion of the cell cycle. We propose that the rate of rRNA production is an important regulatory event in the cell cycle.

The activity of intestinal enzymes (dipeptidase, monoglyceride lipase, amylase, invertase) was studied in intestinal mucosa homogenates from different parts of the small intestine in adult rats 24, 48, and 72 hours after a single intraperitoneal administration of ethionine in a dose of 500 mg/kg bw. 24 hours after antimetabolite administration there was a decrease in the abrasion of the small intestinal mucosa. The enzyme-forming function of the small intestine appreciably changed throughout the observation period. The activity of invertase, dipeptidase and amylase decreased. The monoglyceride lipase activity remained unchanged. The pattern of amylase and invertase distribution in the small intestine changed at the expense of inhibition of their activity in the duodenum and rise in the distal part.

The O2 dependence of cytochrome oxidase, cellular ATP/ADP ratio, and drug sulfation and glucuronidation were studied in isolated hepatocytes to examine secondary biochemical changes of hypoxia related to decreased cytochrome oxidase function. A decrease in cytochrome oxidase function as well as in cellular ATP/ADP ratio occurred at low O2 concentrations and their O2 dependencies gave p50 values of 3.2 and 2.8 microM O2, respectively. Changes in the ATP/ADP ratio corresponded directly with changes in the oxidation-reduction state of cytochrome oxidase. The p50 value for sulfation of acetaminophen (2.5 microM O2) corresponded with the values for ATP/ADP ratio and cytochrome oxidase. Addition of compounds that lowered cellular ATP/ADP ratio caused a corresponding inhibition of sulfation. Selective use of ethionine, an adenosyl-trapping agent, demonstrated that the O2 dependence of sulfation related directly to a decrease in the absolute ATP concentration. The p50 value for glucuronidation (5.2 microM O2) was higher than that for sulfation, but in spite of this difference, glucuronidation also correlated well with cellular ATP/ADP ratio when this ratio was decreased by either O2 limitation or metabolic inhibitors. These results demonstrate that the predominant limitation of drug sulfation and glucuronidation during hypoxia is due to decreased cytochrome oxidase function.

Radioactive alkylated bases, ribose or phosphate, were never found either in acid and alkaline hydrolysates of polyribonucleotides or in alkaline hydrolysates of DNA after incubation with 14C-dimethylnitrosamine (DMNA) in a microsomal system. Two radioactive compounds, which were co-chromatographed with methylamine and N-methylhydrazine, respectively, on column, paper, and thin-layer, were always detected. They differed from the compound derived from 7-methylguanosine after the alkali-mediated fission of the imidazole ring in its molecule. The in vitro system employed well represents the in vivo situation (7-methylguanine which is liberated from DNA after acid hydrolysis); however, it has given results which do not agree with the generally-accepted mechanism of DMNA alkylation at the N-7 position of guanine.

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet for 24 h develop hemorrhagic pancreatic necrosis with a 5-day mortality rate of approximately 50%. At the end of the diet administration, the in vivo discharge of digestive enzymes is blocked, images of exocytosis and luminal membrane recycling disappear, and zymogen granules accumulate within acinar cells. The general ultrastructure, however, remains well preserved, protein synthesis is normal, and intracellular transport of secretory proteins is only slightly retarded. Thus, the CDE diet does not affect the general phenomenon of membrane fusion-fission but specifically inhibits that associated with exocytosis. Twenty-four hours after withdrawal of the CDE diet, discharge of zymogen granules into lysosomes (crinophagy) can be observed, and, 24 h latr, autophagocytosis is noted. Finally, shortly before the onset of pancreatic necrosis, cells of nearly normal appearance are noted to be scattered among cells showing varying degrees of lesion up to complete disruption. Thus, the CDE-induced pancreatic necrosis results from a sequence of events: blockade of exocytosis evolves to crinophagy and autophagy, which might lead to lysosomal activation of zymogens.

1. Ethionine, an analogue of methionine, is known to cause a number of serious changes. In this study, its influence on the NAD + NADH2 content and on the activity of the poly(ADPR)synthetase in rat liver has been investigated. 2. DL-Ethionine decreases the content of NAD + NADH2 in adrenalectomized, but not in normal animals. 3. After injection of nicotinamide the NAD + NADH2 content in the liver of adrenalectomized rats is much higher than in that of normal ones. 4. DL-Ethionine diminishes the NAD synthesis caused by nicotinamide in adrenalectomized animals and increases it in normal ones. DL-Tryptophan induces only a slight NAD synthesis, which is reduced in adrenalectomized animals by thionine. 5. 1-Methylnicotinamide slightly decreases the NAD concentration in normal animals, but increases it considerably in adrenalectomized ones. DL-ethionine reduces the NAD + NADH content in adrenalectomized animals in the presence of 1-methylnicotinamide. 6. The activity of poly(ADPR)synthetase of in-vitro tested nuclei is slightly increased after treatment with DL-ethionine.

The diglyceride species of phosphatidylcholines from livers of male rats after treatment of the animals with ethionine (1 mg/g divided among 4 doses 2 hr apart), phenobarbital (80 mg/kg each day for 3 days), or a combination of the two drugs were determined using gas chromatography. Ethionine treatment greatly elevated the diene species (significant at the 0.005 level for 34:2 and 0.001 level for 36:2). Phenobarbital treatment had no significant effect on the quantity of 34:2 but slightly increased that of 36:2 (significant at the 0.05 level). Both drugs caused relative decreases in the quantities of 38:4 (significant at the 0.001 level for ethionine and at the 0.01 level for phenobarbital). Ethionine decreased the content of 36:4 (significant at the 0.01 level) while phenobarbital treatment did not produce a significant effect on this fraction. Thus, while ethionine produced marked effects on the quantities of the various molecular species, the effects of phenobarbital were less dramatic. Combined treatment with both drugs generally produced levels of species similar to those produced by ethionine alone.

Varying amounts of DL-, L- or D-ethionine were administered intravenously to sheep, either as a continuous infusion, usually over 2 days, or as a single injection. Groups of sucking mice and rats, in their first cycle of hair growth, were given subcutaneous injections of DL-ethionine at several dose rates. Ethionine was a potent inhibitor of wool growth in sheep; the L- and D-isomers appeared equally effective. An infusion of 20 mg/kg DL-ethionine (c. 50 mg/kg0.75) given at a daily rate of 10 mg/kg for 2 days, or an injection of 40 mg/kg DL-ethionine (c. 100 mg/kg0.75), were sufficient to cause the growth of very weak wool and allow the fleece to be readily removed by hand within 3 weeks after dosing. The inhibition of wool growth was usually associated with a concentration of ethionine in blood plasma, during intravenous infusion, of 10 mumol/l or higher. An infusion of DL-ethionine at a daily rate of 1 mg/kg for 12 days caused the growth of weak fibres and substantially reduced both length growth rate and diameter of fibres. The toxicity of ethionine to sheep was dependent on the total dose and the duration of administration. An infusion of 40 mg/kg (20 mg/kg daily for 2 days) produced severe effects, but the sheep recovered; a dose of 14 mg/kg (2 mg/kg daily for 7 days) was lethal. The effects of ethionine on wool growth were reduced or prevented by the concurrent infusion of methionine (10-15 mol/mol ethionine). Doses of DL-ethionine as high as 460 mg/kg0.75 failed to cause hair loss in sucking mice. While body growth was severely retarded at this dose, no deaths occurred. Likewise, DL-ethionine failed to cause hair loss in sucking rats, but was lethal to some rats at a dose of 360 mg/kg0.75.

The activities of an endogenous inhibitor and of a stimulator of cell proliferation were assayed in the livers of sham-operated (SO) or partially hepatectomized (PH) adult rats; rats fed a choline-supplemented (CS) or a choline-devoid (CD) diet; the same diets followed by acute CCl4 intoxication; the same diets supplemented with phenobarbital (PHB); or a CD diet containing DL-ethionine (ETH). The inhibitor and the stimulator were semipurified by fractional ethanol precipitation of a liver cytosolic fraction, and their activities were assessed by means of bioassays in vitro. The livers of SO rats and of rats fed the CS diet contained only inhibitor activity. Following PH, a CD diet, or CCl4 intoxication the inhibitor activity was suppressed, and there was a simultaneous appearance of a stimulator activity. Thus, PH, a CD diet, and CCl4 intoxication cause similar cellular (loss and regeneration) and humoral-homeostatic changes in adult rat livers. We propose that these changes constitute a basic attribute of the mechanism whereby the three conditions affect similarly hepatocarcinogenesis in the rat, especially in the case of a CD diet, because the changes it induces are chronic rather than acute. PHB, another promoter of chemical hepatocarcinogenesis, affected neither the inhibitor nor the stimulator activity. Thus, PHB seems to be acting by a different mechanism than that of the other three agents. ETH did not modify the shift in the balance of the growth-modulating factors induced by a plain CD diet. This shift may account for the marked stimulation of carcinogen-induced oval cell proliferation exerted by a CD diet. The significance of these results is discussed in the context of known effects of a CD diet and of PHB on hepatocarcinogenesis in rats.

A number of sex-related differences in the metabolism of drugs, steroids and xenobiotics have been reported in studies on rats. Generally, male rats tend to metabolize these compounds more efficiently than females. In the studies presented here, male and female rats were fasted for 24-48 hr, and the effects of fasting on total hepatic microsomal cytochrome P-450 were examined. Hepatic cytochrome P-450, as determined by CO difference spectra, was increased significantly as a percentage of control in microsomes from fasted female rats when compared to fasted male rats. Cytochrome P-450 concentration increased from 0.57 +/- 0.07 nmole/mg protein to 0.99 +/- 0.08 nmole/mg protein following a 24-hr fast. In male rats, cytochrome P-450 levels were essentially unaffected by the 24-hr fast. Cytochrome b5 concentration was not altered by fasting. When female rats were fasted for 24 hr and refed, cytochrome P-450 levels were not significantly different from cytochrome P-450 levels in continuously fed animals. Treatment of fasted female rats with the protein synthesis inhibitor ethionine, or the RNA synthesis inhibitor actinomycin D, prevented the induction of cytochrome P-450 in the fasting animal. Cytochrome P-450 concentration in fed animals was not affected significantly by either inhibitor. Induction of cytochrome P-450 by phenobarbital (PB) and 3-methylcholanthrene (MC) under fed and fasted conditions was also investigated in male and female rats. Xenobiotic-induced cytochrome P-450 concentration was significantly higher in fasted female hepatic microsomes when compared to microsomes from fed female rats. Fasting did not significantly affect xenobiotic-induced cytochrome P-450 in male rats. Our results suggest that fasting in female rats results in an increase in cytochrome P-450 which is dependent upon synthesis of RNA and protein.

Ethionine causes a decrease in the amount of rough endoplasmic reticulum in rat liver, the effect being greater in female than in male rats. Rough endoplasmic reticulum isolated from rat liver 24 hr after ethionine injection and stripped of its ribosomes partially lost its in vitro ribosome binding capacity. However, no differences were detected between the binding affinities of ribosomes, isolated from either untreated animals or intoxicated rats, to stripped rough membranes derived from normal rats. Structural changes occur in the rough endoplasmic reticulum of the ethionine treated rats, while the ribosomes are still bound to the membrane.

1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter.

The influence of various experimental conditions such as partial hepatectomy, administration of hepatotoxic drugs such as carbontetrachloride or ethionine, and starvation on the ulcerogenic activity of indomethacin at intestinal level has been studied in the rat. While lesions produced by oral administration of high doses of indomethacin are reduced by starvation, the incidence of ulcers was significantly increased by partial hepatectomy and by hepatotoxic drugs reaching the maximum of severity (intestinal perforation) in partially hepatectomized animals. These findings suggest that ulcerogenic activity of indomethacin is strictly correlated to the drug metabolising capacity of the liver.

Ethionine, the hepatocarcinogenic antimetabolite of methionine, was fed to rats in carcinogenic doses for 1-10 weeks. Levels of 5-methyldeoxycytidine (5-MC) in nuclear DNA and total cellular levels of S-adenosylmethionine (AdoMet) and S-adenosylethionine (AdoEt) were determined at 1, 5 and 10 weeks in livers of control and ethionine-treated animals. The percentage of deoxycytidine residues modified to 5-MC in hepatic DNA of ethionine-fed animals was the same as that in the control animals at 1 week but was 3.6% and 7.6% lower than that observed in control animals at 5 and 10 weeks, respectively. Significant levels of AdoEt, a DNA methylase inhibitor, as well as decreases in the levels of AdoMet were also observed in the livers of ethionine-fed animals. In a second study, the levels of 5-MC, AdoMet and AdoEt were determined in the pancreas, kidneys, testes and thymus of control rats and rats fed ethionine for 10 weeks. Only the testes, an organ known to be susceptible to the toxic effects of ethionine, showed a significant (p less than 0.02) decrease in 5-MC in response to ethionine feeding. AdoEt was present in all tissues studied, except thymus, but at lower levels than those observed in the liver. These results demonstrate that ethionine administration alone under conditions which cause tumors is sufficient for the production of hypomethylated DNA in the target organ and one extrahepatic tissue studied. Hypomethylation of hepatic DNA would appear to result from the accumulation of AdoEt coupled with the decreased levels of AdoMet.

A cell-free protein-synthesizing system active in initiation of translation of both endogenous mRNA and exogenous mRNA has been obtained from postmitochondrial supernatant (S-12) of the liver of ethionine-treated rats by adding reticulocyte ribosomal extract as a source of initiation factor. Formation of polysomes in the course of protein synthesis in vitro has also been demonstrated. Homogenization of the liver in the presence of 50 microM hemin stabilizes the initiation activity of S-12 fraction, which otherwise decays rapidly even at 0 degrees C. The mechanism of inhibition of protein synthesis by ethionine is discussed in view of these results.

The methionine analog, L-ethionine, induces morphological and biochemical changes in cultured HL-60 cells which are indicative of myeloid maturation. After 3 to 5 days of growth in the presence of L-ethionine, the majority of cells have enhanced phagocytic ability. The percentage of cells in the culture which bear complement receptors and which can respond to 12-O-tetradecanoylphorbol-13-acetate with respiratory burst activity increases more than 3-fold. Since the cells fail to become adherent and lose nonspecific esterase activity, we conclude that L-ethionine, like dimethyl sulfoxide, induces granulocytic differentiation of HL-60 cells.

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.

Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids.