Formation of S-adenosylethionine in liver of rats chronically fed with DL-ethionine.

The early changes in the metabolism of L-ethionine were examined in rats preexposed to chronic administration of DL-ethionine. The capacity of liver to accumulate S-adenosylethionine after a single injection of L-ethionine decreases rapidly from the onset of the carcinogenic regimen. This drop is caused by diminished S-adenosylethionine synthesis, a consequence of lower activity of the ATP-L-methionine adenosyltransferase. This change is accompanied by the rapid increase of the concentration of free ethionine and ethionine sulfoxide. The concentration of hepatic ATP depends in the control animals on the L-ethionine dose and is inversely related to the S-adenosylethionine concentration, but in DL-ethionine-pretreated rats it becomes gradually independent of the L-ethionine dose. The alterations in L-ethionine metabolism observed are not attributed to the change in the ratio of hepatocytes to oval cells but rather to the functional alterations of hepatocytes.

Early alteration in liver of rats fed 2-fluorenylacetamide investigated by L-ethionine probe.

The metabolic activity of liver of rats fed a diet containing 0.03% 2-N-fluorenylacetamide (FAA) was investigated using the probe of L-[ethyl-14C]ethionine (613 mumol L-E/100 g body wt.). Shortly after the onset of the carcinogenic regimen, the capacity of liver to accumulate S-adenosylethionine (SAE) began to decline, reaching its minimum (30% of the concentration in control rats) within 3 weeks. This decreased capacity to accumulate SAE results from the FAA-induced decrease in activity of ATP-L-methionine adenosyltransferase. The concentration of hepatic ATP assayed without L-ethionine (L-E) probe also declined during the first 2 weeks of the carcinogenic regimen, but then increased, attaining the normal values within 2 more weeks. Administering the L-E probe to the FAA-fed rats produced an even greater drop in hepatic ATP concentration during the first 2 weeks; however from the third week on, the L-E dose produced no depressing effect, despite the SAE accumulation remaining at its same depressed levels and, therefore, trapping the same amount of ATP as in the previous weeks. The results show that the modification of L-E metabolism and of ATP turnover, observed previously in DL-E fed rats, need not be specific for the carcinogen fed and can occur even when the carcinogens are metabolized by different enzymatic systems.

The synthesis of the adenosyl-moiety of S-adenosylethionine in liver of rats fed DL-ethionine.

The synthesis of the adenosyl-moiety of S-adenosylethionine from glycine was studied in normal rats and rats fed a diet containing 0.30% DL-ethionine for three weeks, using a L-ethionine probe. The liver of rats, pretreated by DL-ethionine feeding, incorporated--after injection of 20 mg L-ethionine/100 g body wt.--six times more C-14 from glycine-2-14C into the adenosyl-moiety of SAE than normal rats. Further increasing of doses up to 100 mg/100 g body wt. did not substantially change the incorporation in pre-exposed rats while in normal rats an almost complete inhibition of the incorporation was observed. The liver cells exposed to a chronic effect of DL-ethionine are able, after higher demands for ATP, to increase the ATP generation more effectively than normal cells. This adaptation to the toxic effect of ethionine represents one of the first alterations of the metabolic functions of liver parenchyme in the course of hepatic DL-ethionine cancerogenesis.

The effect of methionine on the progression of hepatocellular carcinoma induced by ethionine.

Female Wistar rats were fed a semisynthetic diet containing 0.3% DL-ethionine for 52 weeks and, most of them bearing hepatocellular carcinomas, were switched to a basal diet for one week and then received 80 mg DL-methionine per rat (cir. 25 mg/100 g body wt) by gavage once per day, five times a week, over a period of 13 weeks. Methionine treatment significantly affected progression of hepatic tumors induced by DL-ethionine administration. The frequency of rats with hepatocellular carcinomas in the methionine treated group was significantly lower than that in control groups. It therefore appears that methionine treatment resulted in a remission of the malignant process.

Metabolism of N-acetyl-L-ethionine sulfoxide in rats.

N-Acetyl-L-ethionine sulfoxide, the main urinary metabolic product of L-ethionine in rats, is produced and excreted by hepatocytes, as shown by its identification in liver extracts of rats administered L-ethionine and in the cultivation medium from primary culture of rat hepatocytes incubated with L-[ethyl-1-14C] ethionine. The metabolic fate of N-acetyl-L[ethyl-1-14C] ethionine isolated from the urine of rats injected with L-ethionine was examined in rats in vivo. This compound can be metabolized, to a small extent, into the usual metabolic products of L-ethionine: S-adenosylethionine and, to an increased extent to CO2.

The dose-related metabolism of L-ethionine in acute experiments with rats.

A method is described for a simple column chromatographic determination of N-acetylethionine, N-acetylethionine sulfoxide, and alpha-keto-gamma-ethiolbutyric acid on AG 1. With this analytical method and with chromatography on AG 50W, the urinary excretion pattern of ethionine, composed of all the aforementioned compounds, ethionine sulfoxide and S-adenosylethionine was studied in acute experiments in female rats as a function of the dose of L-[ethyl-1-14C]ethionine. Whereas N-acetylethionine sulfoxide is the major urinary metabolite at low ethionine doses, at higher doses increased amounts of unchanged ethionine and ethionine sulfoxide are found and account for the major portion of the administered dose at 460-613 mumol (75-100 mg)/100 g body wt. The urinary excretion pattern of S-adenosylethionine shows a close relationship to the concentration pattern of this metabolite in the kidney. The extent of t-RNA ethylation and the amount of carbon dioxide formed from the ethyl group of ethionine peak at doses of 77 (12.5) and 306 mumol (50 mg)/100 g body wt respectively, and do not increase further at higher doses.

In vivo D-ethionine inversion and its inhibition.

The quantitative study of the inversion of D-ethionine into L-ethionine in vivo, measured in the liver of rats by the formation of S-adenosylethionine from L-ethionine, demonstrates high efficiency of this conversion when lower doses of D-ethionine are used. At higher doses (> 25 mg (80 mumol)/100 g body wt) the accumulation of S-adenosylethionine is retarded. It is suggested that this decrease of S-adenosylethionine formation is due to an unknown as yet effect of D-ethionine on the L-ethionine metabolism. At these higher doses, the alternative assay of the inversion, based on determining the dilution of radioactive L-ethionine probes by inverted D-ethionine, demonstrates considerably higher inversion, supporting the previous assumption. The inversion can be inhibited by administering D-ethionine simultaneously with either kojic acid or sodium benzoate, both inhibitors of D-amino acid oxidase. Sodium benzoate administration is tolerated very well by rats and therefore may be used in chronic experiments.

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.

The metabolism of L-ethionine in rats fed alpha-naphthyl isothiocyanate.

L-[ethyl-2-14C] ethionine was administered in a single dose to rats fed a diet containing 0.10% alpha-naththyl isothiocyanate for a period of time between 6 and 99 days. Ethionine metabolites excreted into urine were investigated as a function of induced pathologic changes in the liver. A short-time exposure (6 and 14 days) to alpha-naththyl isothiocyanate increased the excretion of free ethionine and S-adenosylethionine at the expense of total ethionine sulfoxide. With the extended feeding period the excretion of total ethionine sulfoxide became normalized while the excretion of free ethionine and S-adenosylethionine decreased. The acetylated fraction of ethionine sulfoxide decreased rapidly and reached a minimum in 14 days of feeding. This fraction subsequently started to increase, but did not reach the values obtained in normal rats.

The effect of cupric acetate on ethionine metabolism.

The addition of cupric acetate, a potent inhibitor of ethionine carcinogenesis, to a diet containing ethionine increased the ethionine toxicity. The concentration of S-adenosylethionine in liver was found to be significantly higher when compared to animals fed only ethionine in the diet. Ethionine forms a complex(es) with cupric acetate that is insoluble at a pH higher than 4; however, this complex can be solubilized at a low pH. Ethionine, if administered p.o. in the form of this complex, was absorbed from the intestinal lumen in the same order of magnitude as when administered alone; however, as the body weight increased over 200 g, the portion of absorbed ethionine decreased. The absorption of ethionine bound in the complex was completed within 16 hr compared to 2 hr for free ethionine. This time delay was accompanied by a shift in the concentration maximum of ethionine metabolities in the liver form 8 to 24 hr. When ethionine was administered alone, it was metabolized in the intestinal lumen as demonstrated by the analysis of the soluble intestinal contents; the presence of cupric acetate inhibited this process. The chromatographic analysis of ethionine metabolites in urine of rats treated by the complex revealed an increased excretion of ethionine sulfoxide and other ethionine metabolities at the expense of N-acetylethionine sulfoxide. The increased concentration of S-adenosylethionine in the liver in chronic experiments may be, at least partly, a result of a diminished capacity of the rat to detoxify (acetylate) ethionine sulfoxide, which is considered the main reserve pool of ethionine for the maintenance of a high level of S-adenosylethionine.

The metabolism of ethionine in rats.

The L-[ethyl-1-14C]ethionine metabolites soluble in trichloroacetic acid were studied in rats by the use of column chromatography. After p.o. application of ethionine, its absorption from intestinal lumen was rapid and was complete in less than 2 hr. Any unabsorbed ethionine was later excreted in the feces. During the passage through the gastrointestinal tract, a portion of ethionine was metabolized. The chemical nature and biological significance of these metabolites is not yet known. The fate of absorbed ethionine was investigated in the small intestine, liver, blood, kidney, and urine as a function of time after application. A great part of ethionine was quickly oxidized to ethionine sulfoxide. In liver and kidney, the concentration of ethionine sulfoxide was higher than that of free ethionine. In all organs, the presence of N-acetylethionine sulfoxide was also demonstrated. Ethionine sulfoxide can be reduced and N-acetylethionine can be deacetylated in vivo as demonstrated by the formation of S-adenosylethionine from ethionine sulfoxide and N-acetylethionine. In urine, 4 main components were observed: N-acetylethionine sulfoxide, S-adenosylethionine, ethionine sulfoxide, and free ethionine. Some minor components, as yet unidentified, were also present in the urine and in different organs. The probable site of origin of urinary S-adenosylethionine is the kidney.