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Ethionine intoxication in nonfasted rats leads to a rapid change in the levels of poly(A)+ RNA. Prolonged fasting causes a decrease in poly(A)+ RNA which is not aggravated further by ethionine. The size distribution of poly(A) tracts at the 3' end of mRNA is not affected by ethionine. However, ethionine induces a change in the metabolism of the poly(A) sequence relative to the remainder of the mRNA.

The effects of a potent cholecystokinin (CCK) receptor antagonist, L-364,718, on two forms of experimental acute pancreatitis in mice were evaluated. The antagonist prevented the hyperamylasemia, pancreatic edema, and acinar cell vacuolization that followed administration of a supramaximally stimulating dose of the cholecystokinin analogue cerulein. In contrast, the same dose of L-364,718 (1 mg/kg/6 h) and an even higher dose (10 mg/kg/6 h) failed to prevent the hyperamylasemia, acinar cell necrosis, and mortality that followed administration of a choline-deficient ethionine-supplemented diet. These observations are at variance with those previously reported to follow administration of the relatively weak cholecystokinin antagonist proglumide (Niederau C et al. J Clin Invest 1986;78:1056-63). The observations reported in this communication suggest that cholecystokinin does not play an important role in diet-induced pancreatitis and that CCK receptor antagonists are unlikely to be of benefit in the treatment of clinical acute pancreatitis.

For pancreatic islet transplantation to become clinically applicable, chemical agents are needed which cause selective morphological alterations in the exocrine part of the organ within a few hours after application. DL-ethionine (Aet), cerulein (Cd), seleno-DL-methionine (SeMet) and seleno-DL-ethionine (SeAet) were given to rats during various time periods. At 4 h after i.p. injection, 40 mg/kg SeMet caused the greatest selective morphological alterations in the exocrine pancreas. Acinar structure disappeared, the exocrine cells were abnormal and their nuclei had various dimensions and forms. There were, however, no alterations in the islet cells. At this time, neither Aet, Cd, nor SeAet produced similar changes. SeMet was the most toxic substance and was taken up in the shortest time and in the greatest amount. This selective destructive SeMet effect allows better isolation of the islets from exocrine tissue thereby increasing islet yield and decreasing transplantation volume. Better islet purification may prevent an early rejection.

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet rapidly develop acute hemorrhagic pancreatitis. We have observed that pancreatic acini prepared from these mice are unable to secrete amylase in response to addition of the cholinergic agonist carbachol, although they retain the ability to secrete amylase in response to the Ca2+ ionophore A23187. The CDE diet does not alter the binding characteristics (Kd or the maximal number of binding sites) for muscarinic cholinergic receptors as tested using the antagonist [3H]N-methylscopolamine nor the competition for this binding by carbachol. Addition of carbachol to acini prepared from mice fed the CDE diet does not result in as marked an increase in cytosolic free Ca2+ levels as that noted in control samples (evaluated using quin2 fluorescence). These observations indicate that the CDE diet interferes with stimulus-secretion coupling in mouse pancreatic acini at a step subsequent to hormone-receptor binding and prior to Ca2+ release. This conclusion is confirmed by our finding that the hormone-stimulated generation of [3H]inositol phosphates (inositol trisphosphate, inositol bisphosphate, and inositol monophosphate) from acini labeled with [3H]myoinositol is markedly reduced in acini prepared from mice fed the CDE diet. This reduction is not due to a decrease in phosphatidylinositol-4,5-bisphosphate. This communication represents the first report of a system in which a blockade of inositol phosphate generation can be related to a physiologic defect and pathologic lesion.

The chronic administration of ethionine, an analogue of methionine and alkylating hepatocarcinogen, to rats resulted in the decrease of hepatic drug metabolizing enzyme activities and cytochrome P-450 content. In contrast, the activity of hepatic heme oxygenase, a first enzyme of heme degradative pathway, was increased following ethionine administration. delta-Aminolevulinic acid (ALA) synthetase, a key enzyme of heme biosynthesis pathway, was also increased except during 9 to 15 weeks of the experimental period. Changes of cytochrome P-450 content and heme oxygenase activity were proportional to the carcinogen provided period. Further, ethionine induced neoplastic nodules showed more profound changes in heme oxygenase, ALA synthetase and drug metabolizing enzyme activities than the surrounding liver. Similar changes in the enzyme activities were also obtained by a single administration of ethionine to rats and these effects were dependent upon dose levels of the carcinogen except ALA synthetase activity. These results revealed that ethionine is a mild inducer of hepatic heme oxygenase. It is thus likely that induction of heme oxygenase produced by ethionine could lead to the decrease of cytochrome P-450 and drug metabolizing enzyme activities in the liver. The decrease of heme availability due to induction of heme oxygenase by ethionine may be one of the explanations for the decrease of cytochrome P-450 content under the experimental conditions.

We have confirmed the finding by Rajalakshmi that transfer RNA (tRNA) from livers of ethionine-treated rats can act as a substrate for homologous tRNA-methylating enzymes in vitro. This methyl-deficient tRNA from liver can be methylated in vitro by enzymes from normal or ethionine-treated rats. The in vitro inhibition of tRNA methylation that follows ethionine treatment can be at least partially relieved in vitro. The liver extracts from ethionine-treated animals contained a low-molecular-weight inhibitor of tRNA methylation. Dialysis of enzyme preparations from ethionine-treated, but not control, rats resulted in large increases in tRNA methylase activity, with either Escherichia coli or homologous tRNA's as substrate. Furthermore, the tRNA methylase activity of control rat liver enzyme extracts was greatly depressed by dialysate from liver homogenates of ethionine-treated rats. After 5 days of ethionine administration the liver tRNA methylase activities were significantly higher than those of control preparations despite the continued presence of the dialyzable inhibitor(s). The liver tRNA's from these animals were poorer methyl acceptors than those from 3-day-treated rats, although still better than tRNA's from untreated rats. These observations have been interpreted to indicate that ethionine causes the accumulation in the liver of inhibitors of tRNA methylation. Early in the course of ethionine administration, methyl-deficient tRNA can be isolated. When the period of ethionine treatment is extended, the organism attempts to maintain homeostasis by production of increased amounts of tRNA-methylating enzymes. The increased quantities of these enzymes are able to overcome, at least partially, the effects of the inhibitors and to decrease the extent to which methyl-deficient tRNA is produced.

Starting with a relaxed (relA) strain, mutants with reduced activity of adenosine triphosphate:L-methionine S-adenosyl transferase (EC 2.5.1.6; SAM synthetase) were isolated in Bacillus subtilis. One such mutant (gene symbol metE1) had only 3% of the normal SAM synthetase activity but grew almost as well as the parent strain. Another mutant was isolated (gene symbol spdC1) as being able to sporulate continually at a high frequency; it had one-half the normal SAM synthetase activity at 33 degrees C. Both mutants continually and spontaneously entered spore development at a higher frequency than the parent strain in a medium containing excess glucose, ammonium ions, and phosphate. Sporulation was prevented by a high concentration of SAM (1 mM or more) or by the combination of adenosine and methionine (0.5 mM or more each), both of which are precursors of SAM. In contrast to this continual increase in the spore titer, addition of decoyinine, an inhibitor of GMP synthetase, rapidly initiated massive sporulation. Various amino acid analogs also induced sporulation in the relA strain, the methionine analogs ethionine and selenomethionine being most effective.

In the present study, the nature, proportions and distribution of methylated amino acids in ribosomal proteins from Escherichia coli grown in the presence of ethionine and from mutant prm 1 were studied. The undermethylated ribosomes had been labeled by addition in vitro or in vivo of radioactive methyl groups from S-adenosylmethionine or from methionine. The following compounds were identified : N alpha-mono-, di- and trimethylalanines, N epsilon-mono-, di- and trimethyllysines, methylamine and N alpha-trimethylalanyllysine. Except for the latter compound and N-alpha-dimethylalanine, all other derivatives had been previously identified in the literature. It is shown that the dipeptide had been in the past mistaken for N epsilon-monomethyllysine, and arises through incomplete hydrolysis in 24 hrs of the N-terminal peptide bond of protein L11. The results of the present study are discussed in the light of previous work on ribosomal protein methylation by the authors and other workers in the field.

The chemical inducers of murine erythroleukemia cell differentiation, dimethyl sulfoxide, sodium butyrate, and ethionine, elicited conformational changes in the DNA and chromatin of treated cells. The chromatin from dimethyl sulfoxide treated and butyrate-treated cells exhibited circular dichroic spectra different from that of the noninduced control. The molar ellipticity [theta]282.5 in isotonic saline decreased from 4900 deg X cm2 X dmol-1 for control chromatin to 3800 and 3600 deg X cm2 X dmol-1 for dimethyl sulfoxide treated and butyrate-treated chromatin, respectively, while that from ethionine-treated chromatin remained virtually unchanged (5400 deg X cm2 X dmol-1). Increasing the ionic strength to 2.5 or 5 M with NaCl resulted in a substantial, uniform, decrease in molar ellipticity. Thermal denaturation profiles of high molecular weight DNA prepared from each of the inducer-treated cells showed a pronounced hyperchromic shift but no change in Tm when compared to control DNA. Circular dichroic spectra of the DNA indicated a decrease in ellipticity [theta]277 from 9600 deg X cm2 X dmol-1 to 8900, 8300, and 8800 deg X cm2 X dmol-1 for ethionine, dimethyl sulfoxide, and butyrate treated cells, respectively. Treatment of the DNA with 3 M NaCl canceled the UV and CD differences. These measurements indicate an increased stacking of bases or an increased compactness of the DNA from induced cells. Concomitant with specific modifications such as hypomethylation of DNA, the data can be interpreted in terms of conformational changes in chromatin resulting from core histone acetylation.

The acute biochemical effects of ethionine have been well studied in rats but not in mice. These results show that both hepatic RNA and protein synthesis in Swiss mice were inhibited by DL-ethionine. Protein synthesis was inhibited 30 to 40 percent 3 hr after 2500 mg/kg DL-ethionine while RNA synthesis was inhibited 80 to 90 percent 3 hr after 625 mg/kg DL-ethionine. Thus ethionine was a far more potent inhibitor of RNA synthesis than of protein synthesis. There was no sex difference in either of these responses. While both stereoisomers were active, the L isomer was a more potent inhibitor of RNA synthesis than was the D isomer. In male mice, 3 hr after 20 mg/kg L-ethionine, RNA synthesis was inhibited 80%, while after D-ethionine it was inhibited only 51%.

L-Methionine induced production of ethylene by Saccharomyces cerevisiae growing in lactate medium. The production induced by L-methionine was inhibited by pyruvate, and elevated by glucose. Labeled ethylene was produced when L-[U-14C]methionine, but not [U-14C]glucose, was fed to the yeast. The mutant S. cerevisiae G1332 (ade-, met-) did not produce significant amounts of ethylene unless L-methionine was added. Thus L-methionine acts as a precursor of ethylene in S. cerevisiae. The role of glucose appears to be other than as a precursor.

Transplantation of adult rat pancreatic islet tissue as a free graft requires the separation of islet from exocrine tissue to avoid host injury or graft destruction by digestive enzymes. The poor yield from islet isolation techniques currently necessitates the use of multiple donors to ameliorate diabetes in a single recipient. DL-ethionine (DLE) is an agent selectively toxic to the exocrine pancreas. We examined the effect of DLE administration on pancreatic digestive enzyme content and islet mass in adult Lewis rats and the ability of such pancreatic tissue dispersed by collagenase digestion without specific islet isolation to ameliorate diabetes when transplanted to the portal vein of syngeneic rats with streptozotocin induced diabetes. Rats fed normal chow supplemented with 0.5% DLE for 14-20 days showed a logarithmic loss of pancreatic mass. Total pancreatic amylase content declined to 0.3 + 0.1 mg, less than 3% of control values (14.3 +/- 1.0 mg). Total insulin content in DLE treated rats was 87 +/- 8 microg, not significantly different from control rats (101 +/- 7 microg). Histological examination confirmed the selective atrophy of exocrine tissue in DLE treated rats. Fresh pancreatic tissue prepared from a single DLE treated donor ameliorated diabetes 75% of the time when transplanted to one or two recipients and 65% of the time when divided between three of four recipients. Tissue prepared from a single DLE treated donor and stored for 24-48 hours ameliorated diabetes 91% of the time when divided between one or two recipients. Only four of 31 diabetic rats transplanted with fresh pancreatic tissue from untreated adult donors became normoglycemic. Pretreatment of adult rats with DLE induces selective exocrine atrophy, permits dispersed pancreatic tissue from a single donor to ameliorate experimental diabetes in up to four recipients, and allows tissue to be preserved by culture for up to 48 hours without specific islet isolation.

The two pathways for the biosynthesis of phosphatidylcholine, by way of phosphocholine and by methylation of phosphatidylethanolamine, in wild-type yeast (Saccharomyces cerevisiae) and in the yeast mutant GL7 have been compared. The mutant requires for growth a sterol, unsaturated fatty acids, and methionine. The uptake of labeled choline or labeled methionine and their conversion to phosphatidylcholine were determined in both cell types. The activities of the major enzymes for both pathways were assayed in vitro. We find that the methylation pathway is predominant in both wild-type and mutant cells though the overall activity of the choline pathway is lower in the yeast mutant. The methionine analogue ethionine inhibits the growth of the mutant more strongly than growth of wild-type yeast. Ethionine, while a powerful inhibitor of phosphatidylcholine synthesis by methylation, stimulates the choline pathway in both cell types.

Addition of 10(-2) M L-cysteine, L-cystine, or S-ethyl-L-cysteine to a synthetic medium containing xylose as the sole carbon source did not decrease ochratoxin production by Aspergillus ochraceus. At that concentration, DL-homocysteine thiolactone HC1, DL-cysteine HC1, L-ethionine, S-methyl-L-cysteine, and glutathione (reduced) strongly inhibited ochratoxin production. DL-Homocysteine thiolactone HC1, DL-cysteine HC1, and L-ethionine also strongly inhibited fungal growth. At lower concentrations (10(-3) and 10(-4) M) only L-ethionine decreased the toxin production. Ochratoxin inhibition caused by DL-homocysteine thiolactone HC1, DL-cysteine HC1, and glutathione was observed only in cases where the pH of the media was below 5.0. The inhibition caused by 10(-3) M ethionine was partially prevented by the addition of 10(-3) M methionine but this was not the case after the addition of S-methyl-L-cysteine to the medium.

An hypothesis of carcinogenesis is derived which suggests that carcinogens initially induce derepressions of structional genes of post-translational modifying enzymes. Although the resulting cascade of changes in genic activity would be measureable because of the large number of differentiated cells present in a tissue, only stem cells are vulnerable to the neoplastic array of genic expression by virtue of their repressors being more vulnerable to conformational modification. Furthermore it is derived that there are at least three major classes of repressors and the repressors that are synthesized during embryogenesis are particularly susceptible to derepression mechanisms.

A possible mutagenic and comutagenic activity of ethionine, an analog of the amino acid methionine, was investigated in several mutant strains of E. coli K12. Ethionine was found to act as a weak mutagen only in a mismatch repair deficient mutator strain (mutL) and as a comutagen with 2-aminopurine (2AP) in a wild type E. coli. The latter effect was nor observed in a restriction-deficient strain (r-) nor in a recombination or SOS-deficient recA strain. These effects are interpreted as a consequence of restriction-induced double-strand breaks in hypomethylated E. coli DNA resulting in induction of the SOS mutator effect which generates predominantly mismatch correctable untargeted mutations.

Cytoskeletal and adhesion characteristics of DL-ethionine (CAS: 67-21-0)-transformed rat liver epithelial cells (ETC) were compared with those of nontumorigenic, untreated cells of the same cell line both at the same passage level as ETC and at an early low passage level. ETC and high-passage-level cells (HPC) showed increased cell spreading and prominent actin stress fibers compared to low-passage-level cells (LPC). The number of adhesion plaques per unit cell area was higher for ETC than for LPC. At confluence, fibronectin expression was high for ETC, moderate for HPC, and low for LPC. The observed increases in cell spreading and in actin and fibronectin expression appeared to be associated with transformation of this cell line rather than being specific responses to ethionine treatment. This conclusion is suggested by the fact that HPC, which display preneoplastic markers, are similar in many respects to ETC.

A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.

Six ethionine-resistant (Etr) regulatory mutants of Saccharomycopsis lipolytica Sl/1 overproducing methionine have been isolated. Five of them are also resistant to seleno-methionine. The activity of homocysteine synthase (O-acetyl-L-hormoserine-acetate lyase, adding hydrogen sulfide) is derepressed in these mutants and is not susceptible to the methionine-mediated repression. The pool of free methionine in Etr mutants is enhanced 1.5 to 18 times, and incorporation of 35S into methionine is 1.5 to 50 times higher than that in the wild strain. Neither accumulation of endogenous free methionine in Etr mutants nor the uptake of exogenous methionine is accompanied by an increase in the S-adenosylmethionine pool. This implies compartmentation of methionine metabolism in S. lipolytica.

2--4 mM L-ethionine completely inhibits DNA synthesis in phytohaemagglutinin- or concanavalin A-stimulated lymphocytes even though it does not prevent the morphological changes characteristic of blast formation. Evidence is presented which indicates that complete commitment to DNA synthesis as well as a substantial increase in the rates of RNA and protein synthesis can occur in the presence of ethionine. Ethionine, however, does inhibit methylation of tRNA and prevents mitogen-induced increase in the activity of histone-modifying enzymes. All of these effects of exposure to ethionine are completely reversible. Removal of ethionine after 24 h or more of exposure results in a rapid, synchronous wave of DNA synthesis, an increase in the rate of methylation of RNA and an increase in activity of histone-modifying enzymes.

The radioactivity from 3H-methyl methionine was rapidly incorporated into the surface lipids of Mycobacterium avium. The transmethylation reaction was efficiently inhibited by DL-ethionine, D-norleucine and DL-norleucine. The structure of the outerlayer of the M. avium envelope was profoundly altered in the bacteria treated with DL-norleucine.

DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be prevented by adenine, biotin or lecithin are most probably related to the inhibition of methylation.

Ethanol reversed the ethionine-induced inhibition of 3H-thymidine incoroporation into hepatic DNA in the male and female rat after partial hepatectomy.

Female NMRI mice were fed diets containing l-ethionine (0.1 and 0.3% w/w) and phenylalanine (3% w/w), as well as respective control diets. Ethionine, the S-ethylated analog of methionine, was shown to inhibit phenylalanine hydroxylase in vivo, whereby in vitro kinetics remained unaffected. Treatment with ethionine resulted in fatty liver, reduced ATP content of liver, and alterations in serum amino acid concentrations. In the high dosage ethionine group, for instance, concentrations of Ala, Gly, Ser, Met, and Phe were increased whereas concentrations of Lys, Asp, and Pro were decreased. Applying ethionine together with phenylalanine resulted in hyperphenylalaninemia and phenylketonuria. Feeding phenylalanine alone also led to decreased activity of phenylalanine hydroxylase and increased concentration of Phe in serum. Ethionine only had a minimal effect on body weight gain; however, the hyperphenylalaninemic condition induced by application of the high dosage of ethionine and phenylalanine induced severe loss of body weight. A disturbed protein synthesis and protein phosphorylation might be the underlying mechanism of ethionine-induced suppression of phenylalanine hydroxylase.

Nicotinamide is methylated via S-adenosylmethionine (AdoMet) and excreted in the urine as N1-methylnicotinamide. Since S-adenosylethionine (AdoEth) inhibits nicotinamide methyltransferase, ethionine (0.75 mg/g body wt) was given to rats and its effect on the urinary excretion of N1-methylnicotinamide was examined. Nicotinamide (0.5 mg/g body wt) gave a 13.6-fold increase in the 24-h urine concentration of N1-methylnicotinamide. However, ethionine given 4 h prior to nicotinamide completely prevented this increase by nicotinamide. It appears at ethionine via AdoEth inhibits nicotinamide methyltransferase in vivo and lowers the urine level of N1-methylnicotinamide.

How closely the isolated liver of the rat would simulate the in vivo function of the organ in terms of the metabolic pattern of the compounds such as bromosulphophthalein, p-nitrophenol, hexobarbital, and indocyanine green was investigated. In order to produce tissue with the stimulated function, the animal was pretreated with phenobarbital and, for the reverse purpose, with ethionine. Some of the indices of the function employed herein, such as the appearance pattern of the compound, the rate of biochemical transformation or the biliary excretion, showed that the perfused liver would generally well reflect the in vivo situation. The method with isolated and perfused liver could exclude the participation of other organs and also the influence of the factors unavoidable in an in vivo experiment. Thus, it is suggested that the isolated perfused liver is useful for studying directly the functional level of the organ as drug metabolizing tissue.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The state of mRNA and monomeric ribosomes after the polysome dissociation was studied. The mRNA was selectively labeled with [14C] orotate after a low dose of actinomycin D. Sucrose density gradient centrifugation of Triton X-100-treated cytoplasm revealed an accumulation of heterodisperse radioactive material with very large S values. This material was converted to smaller S values with deoxycholate treatment and was extremely sensitive to mild ribonuclease treatment. Since this material was banded at around 1.43 g/cm3 in CsCl gradient centrifugation and contained RNA with a distribution of S values characteristic of polysomal mRNA, this material was identified as mRNA-containing ribonucleoprotein particles. The monomeric ribosomes were shown to be dissociated into subunits in the presence of 0.5 M KCl, indicating that these lacked nascent polypeptide chains. When the animals were recovered from the ethionine treatment by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appeared to be utilized for the reformation of polysomes.

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [14C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10(4) and 2.1.10(4) were decreased in ethionine-treated rate liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[14C]ethylmaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

Ethionine, a hepatocarcinogen, was administered into rats 24 h before partial hepatectomy and immediately thereafter. Hepatic precursor ribosomal RNA (pre-rRNA) obtained 20 h after the operation of rats injected with ethionine and adenine resulted in methyl deficiency as judged by the incorporation of [3H]methyl group of S-adenosylmethionine into nuclear rRNA by partially purified rRNA methylase. The ethionine and adenine treatment causes methyl deficiency of nuclear rRNA at 2'-hydroxyribose sites of cytidine and uridine, but not at base sites. Although the ethionine and adenine treatment produced no significant change in total hepatic RNA synthesis in vivo assayed by the incorporation of labeled orotate, a one-third increase in nuclear rRNA synthesis as well as a one-third decrease in microsomal rRNA synthesis was found under the treatment. These results suggest that the undermethylation at 2'-hydroxyribose of pre-rRNA in liver nucleus, which is caused by ethionine and adenine administration into rats, causes an inhibition of the processing of nuclear pre-rRNA to cytoplasmic rRNA.

The extent of single-strand nicks in DNA from murine erythroleukemia cells induced to differentiate to hemoglobin synthesis in the presence of the hypomethylating agent ethionine was estimated and compared to those levels in uninduced cells and from cells induced to differentiate upon exposure to dimethylsulfoxide or butyrate ion. Although ethionine has been shown to cause more extensive hypomethylation in the DNA of induced cells than that caused by dimethylsulfoxide or butyrate ion, the frequency of detected single-strand breaks in the DNA of uninduced, control cells was not significantly different from that of cells exposed to any of these inducing chemicals. This data indicates that no correlation exists between DNA hypomethylation and DNA single-strand breaks and that unmethylated CpG loci likely do not operate as specific endonuclease recognition sites or as potential origins of transcription in these mammalian cells.

Compared with the parental strain, a Spirulina platensis mutant that is resistant to ethionine incorporated methionine into protein at a reduced rate, whereas ethionine incorporation was practically nil. The methionyl-tRNA synthetase present in crude extracts from the resistant strain showed a reduced affinity for methionine and ethionine.

DL-Ethionine (0.87 g/kg of body weight) was administered IV to 6 fasted (12 hour) Holstein bull calves (4.5 months old). Fasting, which was continued for an additional 48 hours, caused a body weight loss (10.82% to 11.96%), a mild increase in PCV, and an increase in the serum free fatty acids (1.204 mmole/L, fasted; 0.949 mmole/L, fasted ethionine-treated calves). Ethionine caused a decrease in the total plasma proteins from 6.5 g/dl to 5.5 g/dl and total serum lipids from 493.9 mg/dl to 307.8 mg/dl. The decrease in the serum esterified fats included all the lipid fractions (triglycerides, cholesterol, and phospholipids). The calves' WBC and serum enzymes aspartate aminotransferase and L-alanine aminotransferase remained within normal range.

An assay for nicotinamide methyltransferase which uses an Affi-Gel 601 boronate column to separate substrate from product has been used to study the inhibition of the nicotinamide methylase. S-Adenosylethionine and S-adenosylhomocysteine inhibited the enzyme in a dose dependent fashion. Five times more S-adenosylethionine than S-adenosylhomocysteine was necessary for 50% inhibition of the enzyme. Data from a substrate concentration curve supports a competitive type of inhibition for both inhibitors. It is proposed that this inhibition may alter liver levels of nicotinamide and the urinary excretion of N1-methylnicotinamide.