[CANCER RESEARCH 39, 3935-3941, October 1979] 0008-54 72/79 /0039-OOOOS02.00 Identification of a Transaminative Pathway for Ethionine Catabolism1 Robert D. Steele2 and Norlin J. Benevenga3 Departments of Nutritional Sciences and Meat and Animal Science, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT extensively in recent years in an effort to explain why methio nine is the most toxic of the dietary amino acids (19). Studies The potential for the oxidation of ethionine by a transamina- by Benevenga et al. (7-9, 26) showed that a pathway for tive route was studied in an attempt to elucidate the pathways methionine oxidation independent of S-adenosylmethionine whereby the ethyl carbons of ethionine are converted to carbon formation was operative and that formaldehyde and formate dioxide. Ethionine is transaminated based on the recovery of were 2 intermediates in the oxidation of the methionine methyl a radioactive phenylhydrazone derivative. Carbon 1 of the ethyl group. It has since been shown that a transaminative pathway portion of ethionine is recovered as carbon dioxide when L- [ef/jy/-1-14C]ethionine is incubated in a rat liver homogenate of methionine catabolism represented a major part of the catabolism of methionine in liver homogenate preparations of system. The addition of pyruvate as an amino group acceptor rats and monkeys (27, 37). Methionine was shown to be for transamination stimulated oxidation by more than 10-fold transaminated to its keto acid, a-keto-y-methiolbutyrate, and based on carbon dioxide formation and on recovery of the a- then decarboxylated to 3-methylthiopropionate (37). Rats fed keto acid of ethionine as the phenylhydrazone derivative. The diets with high levels of 3-methylthiopropionate exhibited addition of 3-ethylthiopropionate to the incubations resulted in complete inhibition of 14CO2formation from 10 rriM L-[efhy/-1- growth and food intake depressions and darkened and en 14C]ethionine. The expanded 3-ethylthiopropionate pool was larged spleens (36) similar to those seen in rats fed high levels of methionine (2). In rat liver, 3-methylthiopropionate was found isolated by anion-exchange chromatography to determine if it to be catabolized to methanethiol, hydrogen sulfide, sulfate, had become labeled during the incubation. Gas-liquid chro and carbon dioxide (39). These results suggested that an matography of the isolated products revealed a major radio intermediate(s) produced in this pathway was probably asso active peak with a retention time identical to that of a reference ciated with the toxicity of methionine. sample of 3-ethylthiopropionate. Mass spectral analysis of this The metabolism of ethionine, the carcinogenic analog of radioactive peak obtained from liver homogenate incubations methionine, is not well understood. Ethionine is known to be was identical to the spectra obtained from authentic 3-ethyl activated to an S-adenosyl derivative which has been found to thiopropionate. The same gas Chromatograph peak and mass act as an ethyl group donor in competition with some normal spectra were obtained when a boiled liver homogenate was methylation reactions of S-adenosylmethionine (32). An abnor incubated with i_-[efhy/-1-14C]ethionine and an expanded 3- mal alkylation of nucleic acids by S-adenosylethionine has ethylthiopropionate pool; however, as expected, the peak re been postulated to be involved in the hepatotoxicity of ethionine covered from the gas Chromatograph was not radioactive. and thus to contribute to its carcinogenicity (15). These results indicate that 3-ethylthiopropionate is formed, Little information is available on the oxidation of ethionine to probably as a result of decarboxylation of the a-keto acid of carbon dioxide. Early studies showed that only about 3% of ethionine and is thus an intermediate in ethionine catabolism. the administered dose of L-{efr)y/-1-14C]ethionine was re Rats fed a diet containing 1.5% of 3-ethylthiopropionate ex covered as 14CO2after 24 hr. However, the oxidation of ethio hibited severe depressions in growth and food intake and nine was apparently dose dependent because about 3 times displayed marked neuromuscular abnormalities. Mortality was 40% over the 2-week experimental period. The animal's breath more radioactivity was recovered in CO2 when the amount of ethionine administered was increased 10-fold (15). Stekol ef had an odor that was indistinguishable from ethanethiol. Ex al. (40) have confirmed these findings, and in addition they periments with the liver homogenate system demonstrated the formation of labeled ethanethiol in addition to 14CO2from 10 have found that male rats have a greater capacity to oxidize HIM [ef/iy/-1-'4C]-3-ethylthiopropionate. This pathway appears ethionine than do female rats. Recent studies by Brada ef al. (4) found that about 6% of a p.o. or injected dose of L-[ef/7y/-1 - to account for the majority of ethionine oxidation in a liver 14C]ethionine was recovered as 14CO2after 24 hr. homogenate system. From these studies, it appears that this The enzymatic mechanism for oxidation of the ethionine ethyl oxidative pathway may be intimately involved in the etiology of group to carbon dioxide is presently unknown. However, recent the many biochemical alterations that have been reported after results from our laboratory (27) and others (11,21) suggested ethionine administration. that ethionine may be catabolized by the transaminative route that has been postulated for methionine catabolism. Mitchell INTRODUCTION and Benevenga (27) found ethionine to be a good substrate for The oxidative metabolism of methionine has been studied transamination with pyruvate or a-keto-y-methiolbutyrate in the rat liver homogenate system. When leucine transaminase was 1 This work was supported by funds from the College of Agricultural and Life Sciences and by Grant AM 15227 from the NIH. A preliminary report of this work purified to homogeneity from rat liver mitochondria by Ikeda ef has appeared (40). Department Manuscript 727. al. (21), it was found that ethionine transamination with a- 2 Present address: Department of Nutrition, Rutgers University. New Bruns ketoglutarate was twice that observed with leucine and a- wick, N. J. 08903. 3 To whom requests for reprints should be addressed. ketoglutarate. Similarly, studies with purified preparations of Received April 19. 1979; accepted June 22, 1979. glutamine transaminase from rat kidney showed ethionine also OCTOBER 1979 3935 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. R. D. Steele and N. J. Benevenga to be an excellent substrate for transamination by this enzyme g were used in all experiments. They were housed individually (11). as described above. A commercial laboratory chow and water In the experiments reported in this paper, we show that, in were offered ad libitum. rat liver homogenate preparations, ethionine is transaminated to its a-keto acid derivative and catabolized to 3-ethylthiopro- Tissue Preparation and Incubation Conditions pionic acid which yields ethyl mercaptan and an unknown Rats were killed by decapitation, and the livers were excised, product. In addition, we demonstrate the marked toxicity of 3- placed on ice, and then homogenized in 4 volumes of 0.25 M ethylthiopropionate in rats and speculate that this pathway may sucrose with a Potter-Elvehjem tube fitted with a Teflon pestle. be important with respect to ethionine toxicity. Incubations were carried out in 50-ml Erlenmeyer flasks under a flowing oxygen atmosphere for 30 min at 37° in a shaking MATERIALS AND METHODS water bath. Except where noted, the incubation mixtures con tained the following in a final volume of 5 ml: 2 ml of the 20% Isotopes and Chemicals homogenate; 20 HIM potassium phosphate buffer (pH 7.5); 1 D-{ty-14C]Glucose was purchased from New England Nuclear, mM NAD; 1 mM ATP; 5 HIMMgCI?; and 10 mw labeled substrate. When the substrate was [efhy/-1-'4C]ethionine, 10 mM sodium Boston, Mass., and was used without further purification. L- [efr?y/-1-'4C]Ethionine was purchased from ICN, Irvine, Calif. pyruvate was included as an amino group acceptor for trans Radiochemical purity of the ethionine was determined by chro- amination. Water and 0.5 M sucrose were added to attain an matography of an aliquot on a 0.6- x 125-cm analytical cation- osmotic pressure of 280 mOsmol/liter. Blank incubations were exchange column (18). If necessary, the radioactive com carried out after the reaction mixtures and enzymes were boiled pounds were purified to greater than 98% purity the day before for 1.5 min prior to the addition of the radioactive substrate. an experiment by cation-exchange chromatography on 0.6- x Values reported have been corrected for product formation in 12-cm glass columns packed with 400 mesh Dowex 50-X8 the blank incubations. Incubations were stopped by the addi (sodium form) and then lyophilized as has been described tion of 1 ml of 2 N perchloric acid. Gassing was continued for previously (7). Synthesis of [efhy/-1-14C]-3-ethylthiopropionic 45 min to ensure complete recovery of volatile products. In acid from correspondingly labeled ethionine was accomplished experiments with ethionine as the substrate, the CO2 that by degradation with ninhydrin and oxidation of 3-ethylthiopro- evolved during the incubations was trapped in 3 ml of ethanol- pionaldehyde to 3-ethylthiopropionate by yeast aldehyde de- amine:methyl cellosolve (1:2, v/v). Radioactivity was deter hydrogenase (EC 1.2.1.5). The details of this procedure are mined in the traps by counting aliquots by liquid scintillation reported elsewhere (38). Radiochemical purity of the isotope spectrometry with an organic scintillation fluid (23). Counting used in each experiment was checked by paper chromatog error of the samples was less than 1%, and counting efficiency raphy and was found to be greater than 98% in all cases. of each sample was determined by using an automatic external Nonlabeled 3-ethylthiopropionate was kindly provided by Dr.