Identification of a Transaminative Pathway for Ethionine Catabolism1

[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

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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 37Â° 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. standard. Rudolf Fahnenstich of Degussa Chemical Company, Teterboro, When 3-ethylthiopropionate was the substrate, 2 additional N. J. Purity of this preparation was established by paper and trapping solutions were used to trap any potential volatile sulfur thin-layer chromatography and also by gas-liquid chromatog compounds. In some experiments, the gasses evolved were raphy using the system described below. All other chemicals trapped in 8 ml of a 1% zinc acetate solution for identification were purchased from commercial sources and were of the and determination of hydrogen sulfide by the mÃ©thylÃ¨neblue highest grade available. method described by Siegel (34). Hydrogen sulfide concentra tions were determined from a standard curve that was devel in Vivo Studies oped by using sodium sulfide as a standard. In other experi ments, the gasses produced were first swept through a trap Male Holtzman rats weighing about 75 g were used in these containing 5 ml of a 2% solution of mercuric acetate to remove experiments. They were housed individually in suspended any potential mercaptans before passage through the CO2 cages with wire mesh bottoms at 23Â° in a room with a 12-hr trap. Control experiments with sodium [14C]carbonate release light-dark cycle. Water and diet in agar gel form were available demonstrated that no ÃŒ4CO2wastrapped in the mercuric ace ad libitum. tate traps, and quantitative recovery of 14CO2was obtained in Rats were fed a basal diet of 10% casein for a 3-day the ethanolamine:methyl cellosolve trap. The mercuric acetate adjustment period. The animals were then separated into 2 traps were lyophilized and analyzed for mercaptans by a pro groups of the same average weight and fed either the basal cedure modified from Zieve ef al. (10, 13). Approximately 100 diet or the basal diet supplemented with 1.5% of 3-ethylthio mg of the lyophilized traps were placed in conical glass reaction propionate for 14 days. Supplementation of 3-ethylthiopropio vials. One hundred /il of toluene 3,4-dithiol were added, and nate was made at the expense of the cerelose and cornstarch. the vials were closed with Teflon-lined septa. The reaction vials Animals were weighed daily. After 14 days, all animals were were heated in aluminum heating blocks at 250Â° for 5 min to anesthetized with ether, and blood was drawn by cardiac initiate a flash exchange reaction. The resulting head space puncture for hematocrit and hemoglobin determination (22). gasses produced were sampled with a gas syringe and injected Liver, spleen, kidneys, and heart were removed, blotted, and into the gas Chromatograph for analysis as described below. weighed. Samples inactivated with perchloric acid were centrifuged at In Vitro Studies 15,000 x g for 15 min. The supernatants were neutralized with 6 N potassium hydroxide, centrifuged, lyophilized, and stored Animals and Diets. Male Holtzman rats weighing 150 to 300 at â€”80Â°.In some experiments, the a-keto-y-ethiolbutyrate

3936 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Mammalian Ethionine Metabolism formed from ethionine during the incubations was recovered Table 1 Transamination and oxidation to carbon dioxide of L-{ethyl-l-"C]ethionine: and quantitated as the phenylhydrazone derivative by tech effect of pyruvate and 3-ethylthiopropionate niques described by Mitchell and Benevenga (27). L-[etf7y/-1-14C]Ethionine (10 mM) was incubated for 0.5 hr in flasks, as de scribed under "Materials and Methods." Results are expressed as fimol of ethyl carbon of ethionine recovered in "CO? or ['4C]phenylhydrazone per g of wet liver per hr. The amount of product produced (^mol) was calculated from the Chromatographie Procedures specific activity of the [efhy/-1-'X]ethionine in the incubation. Ion-exchange chromatography of L-[ef/7y/-1-14C]ethionine and its degradation products produced during the incubations AdditionsNone drazone0.75 was performed by using a 0.6- x 34-cm glass column packed Â±0.01a Â±0.40 with 400 mesh Dowex 1-X8 (acetate form) and eluted with a Pyruvate (10 mw) 0.85 Â±0.02 16.38 Â±0.34 Pyruvate0.21a (10 mM) + 3-ethylthiopropionate 0.12 Â±0.01["CJPhenylhy-15.88 Â± waterhydrochloric acid (1 N) constant-volume (500 ml) gra (2.5 mM)"CO20.08 dient with a column flow rate of 0.5 ml/min. The eluate was Mean Â±S.E. of 3 observations. monitored for radioactivity with a Packard flow cell and col lected in a fraction collector. Fractions corresponding to se ate, and even less of the labeled ethyl carbon was recovered lected radioactive areas were combined, neutralized, and ly- as carbon dioxide. However, the addition of 10 mM sodium ophilized for further study. pyruvate to the incubations resulted in a 20-fold increase in Gas chromatography was performed on a Packard Model oxidation of L-[ef/7y/-1-14C]ethionine to carbon dioxide. These 419 gas Chromatograph equipped with dual-flame ionization results are in good agreement with recent studies by Mitchell detectors. Column length and packing and the column, injec and Benevenga (27) who showed that, in addition to the a-keto tion port, and detector temperatures are described in the chart acid of methionine, pyruvate was also an excellent cosubstrate legends. Nitrogen was used as the carrier gas at a flow rate of for ethionine transamination. The effect of other a-keto acids about 35 ml/min. as cosubstrates for the transamination of ethionine in the liver In some experiments, a stream splitter was connected to the homogenate system has not been investigated. column before the detector so that 90% of the effluent was The addition of 2.5 mM 3-ethylthiopropionate to the incuba diverted and collected with a Packard Model 852 gas fraction tions resulted in an 85% decrease in recovery of 14CO2from collector. The inlet tube that led to the fraction collector was L-[efriy/-1 -14C]ethionine; an effect which was similar to but more maintained at the temperature of the detector to prevent pre pronounced than the dilution of 14CO2 production from L- mature condensation of the eluted compounds. The effluent [mef/7y/-'4C]methionine by 3-methylthiopropionate (37). Based was condensed on glass wool-filled cartridges inserted into a on recovery of radioactivity in the phenylhydrazone derivative, turntable which was manually advanced. A well in the turntable the addition of 3-ethylthiopropionate had essentially no effect was filled with dry ice in ethanol to ensure complete conden on transamination of ethionine. sation of the compounds in the effluent onto the glass wool. The effect of 3-ethylthiopropionate on ethionine ethyl carbon The glass wool was expelled from the cartridges into scintilla oxidation to carbon dioxide was studied in more detail. Chart tion vials; water and aqueous scintillation fluid were (6) added, 1 shows the effect of varying levels of 3-ethylthiopropionate on and the radioactivity was determined as described above. L-[ef/7y/-1-14C]ethionine oxidation to 14CO2. Essentially, com When volatile mercaptans were to be collected, the effluent plete blockage of 14CO2 production from ethionine occurred from the fraction collector was collected directly into scintilla with the addition of 10 mM 3-ethylthiopropionate to the liver tion vials containing 4 ml of NCS tissue solubilizer (Amersham/ homogenate incubations. The values shown for CO2 production Searle Corp., Arlington Heights, III.) and counted for radioac from ethionine and the degree of inhibition by 3-ethylthiopro tivity with the organic scintillation fluid described above. pionate are in agreement with the data reported in Table 1. Gas chromatography-mass spectrometry was carried out Because the effect of 3-ethylthiopropionate on ethionine with a Varian 2780 gas Chromatograph connected to a Dupont oxidation could be explained as well by an effect on cellular 21-491 B mass spectrometer. The column length and packing metabolism, its addition on the conversion of glucose carbon and column, injection port, and detector temperatures used (10 mM [U-14C]glucose) to CO2 was studied in the same rat liver are described in the chart legends. Helium was used as a homogenate system. Chart 1 shows that glucose oxidation was carrier gas at 30 ml/min. The analytical system was of all glass not affected by 3-ethylthiopropionate at any concentration construction and used a jet separator. Peaks were detected by studied. These observations indicate that the depression in plots of total ion current versus time, and spectra were taken ethionine oxidation is specifically associated with ethionine manually during the elution of the samples. metabolism and not with general cellular oxidation. The results of these studies indicated that ethionine is quite RESULTS possibly oxidized by a transaminative pathway similar to the one suggested for methionine catabolism (37), and that the The results of initial studies on ethionine ethyl group catab- decrease in recovery of 14CO2from L-[ef/7y/-1-'4C]ethionine by olism in the rat liver homogenate system are shown in Table 1. 3-ethylthiopropionate is due to expansion of an intermediate In a similar system, the recovery of the a-keto acid of methio- metabolite pool. Experiments therefore were conducted with nine as the phenylhydrazone derivative was 54 Â±4% (S.D.) the purpose of isolation of the expanded 3-ethylthiopropionate (1 ). However, since only relative rates of transamination were pool to determine if it becomes labeled during incubation of L- of interest in these studies, no corrections for recovery of the [effty/-1-14C]ethionine in the rat liver homogenate system. phenylhydrazone derivatives were made. With no added cofac- These incubations were performed with 5 mM 3-ethylthiopro tors, little ethionine was transaminated to a-keto-y-ethiolbutyr- pionate as a trapping pool. After a 1-hr incubation, the reac-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. R. D. Steele and N. J. Benevenga tions were stopped with perchloric acid, and the supernatants concentrated under nitrogen and analyzed by gas chromatog- were recovered, neutralized, and lyophilized. Aliquots of con raphy. centrated supernatants from active homogenates and heat- Elution profiles of a standard solution of 3-ethylthiopropio inactivated homogenates were chromatographed on a Dowex nate in ether and of the ether extract from the homogenate 1 column and eluted with a water:hydrochloric acid gradient as incubations are shown in Chart 2. A major peak was eluted at described previously (37). Areas of the elution profile corre approximately 9.5 min in both samples and accounted for sponding to 3-ethylthiopropionate, determined by prior chro- about 80% of the total peak area, excluding the ether solvent matography of [efhy/-1-"'C]-3-ethylthiopropionate, were col peak at the beginning of the elution. The peak that was eluted lected, neutralized, lyophilized, and used to determine the at about 3 min appears to be due to contamination or break radioactivity of the 3-ethylthiopropionate pool. The lyophilized down since it was also observed upon chromatography of the samples were dissolved in acid and extracted with 5 volumes reference sample of 3-ethylthiopropionate. The elution profile of peroxide-free diethyl ether. The ether extract was carefully of standard 3-ethylthiopropionate and that isolated from the incubation were qualitatively and quantitatively identical. 2.5 During gas chromatography of these samples, a portion of the effluent was diverted with a stream splitter so that the distribution of the radioactivity injected onto the column could also be determined. Table 2 shows the distribution of radioac-

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1.5

3- LO

4 8 I2 I6 20 3-ETHYLTHIOPROPIONATE CmM) 24 6 8 IO I2 Chart 1. Effect of increasing concentrations of 3-ethylthiopropionate on the COLUMN RETENTION TIME CMIN) conversion of 10 mM L-{efr>y/-1-"C]ethionme to "CO2 and of 10 rtiM [U-"C]- Chart 2. A, gas chromatography elution profile of radioactive compounds glucose to ÃŒ4CO?inrat liver homogenate incubations. Incubations were carried isolated by aniÃ³n exchange chromatography of supernatants from liver homog out for 30 min at pH 7.5 as described under "Materials and Methods." Each enate incubations with L-(efhy/-1 -"CJethionine. B. elution profile from a reference point represents the mean of 4 observations. Coefficients of variation for each sample of 3-ethylthiopropionate. A 5-ft column packed with 10% SP-1200/1% point were less than 5%. The amount of CO2 produced was calculated from the H3PO4 on 80 to 100 mesh Chromosorb W AW was used with a flame ionization specific activity of the added labeled substrate A. [efny/-1-'4C)ethionine; â€¢[U- detector. Temperature parameters were: column, 150Â°;injection port. 175Â°;and "C|glucose. detector, 180Â°.

Table 2 Distribution of radioactivity recovered during gas-liquid chromatography of isolated metabolites trom rat liver incubations Liver homogenates were incubated with L-tefhy/-1-MC]ethionine as described under "Materials and Methods." The supernatants were treated as described in the text. After a sample was injected into the gas Chromatograph, the column effluent was trapped by using a gas fraction collector, and the fractions were counted for radioactivity. The retention times shown correspond to those detected in Chart 2 The results are reported as the percentage of the total recovered radioactivity recovered during the indicated column elution interval. timesSampleUnknownBlankc0-2 dpm at following column retention of recov- min10.3 min5.8 min7.5 1min68.3 min8.8 erya73.2 Â±1.66 Â±0.9 Â±0.618.9 Â±3.8 Â±1.1 Â±3.8102.3 29.3 Â±0.73-5 16.3 Â±2.26-8 Â±1.89-1 20.8 Â±1.011-1314.7 Â±2.4% Â±3.3

, Total dpm recovered x 100 dpm injected 0 Mean Â±S.E. for 6 observations (unknown) and 3 observations (blank). c The blank samples were prepared from incubations which were boiled for 1.5 min before the addition of labeled substrate

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Mammalian Ethionine Metabolism tivity in injected samples prepared from standard incubations and from blank incubations in which the incubation mixture had been boiled for 1.5 min prior to the addition of L-fefhyM- 14C]ethionine. In incubations conducted with labeled ethionine, about 70% of the radioactivity that was recovered from the column was associated with the peak detected at 9.5 min (Chart 2). The radioactivity recovered from the column when samples from the blank incubations were injected was evenly spread across the elution profile. Because little 3-ethylthiopro- JL pionate would be expected to be produced in the blank, the total amount of radioactivity injected was also only 10% of the amount injected in the other samples. Thus, while the peak at 9.5 min was still detected in about the same relative amount as the test samples, there was no radioactivity associated with it. Attempts to divorce the radioactivity from the peak at 9.5 min 40 by altering gas flows or changing column temperature were not successful. Therefore, production of labeled 3-ethylthiopro- 20 pionate is enzyme dependent. A comparison of the unknown radioactive compound and a JUL 0.5 I.O I.5 2.0 2.5 3.0 reference sample of 3-ethylthiopropionate was also made uti COLUMN RETENTION TIME (MIN) lizing gas chromatography-mass spectrometry. A series of Chart 4. A. gas chromatography of a volatile radioactive compound released from mercuric acetate traps by flash exchange with toluene 3,4-dithiol as de mass spectra were taken during the elution of a reference scribed under "Materials and Methods." 6, elution profile of a reference sample sample of 3-ethylthiopropionate and during the elution of the of ethanethiol. A 12-ft column packed with 30% SE-30 on 80 to 100 mesh samples isolated from the rat liver homogenate incubations. Chromosorb P was used with a flame ionization detector. Temperature parame ters were: column, 75Â°;injection port, 150Â°;detector, 150Â°. Analysis of the spectra obtained from the reference sample indicated that the peak that eluted from the column at 9.5 min was 3-ethylthiopropionate (Chart 3). Spectral analysis also possibility that radioactive ethanethiol could be recovered after incubation of [ef/jy/-1-'4C]-3-ethylthiopropionate was investi showed that the radioactive peak at 9.5 min (Chart 2) recovered from the rat liver homogenate incubations was also 3-ethylthio gated. Incubations were carried out, and the gasses evolved propionate. A number of spectra were taken during the begin were trapped in a mercuric acetate solution. Gas chromatog ning, the middle, and at the end of the radioactive peak eluting raphy of the head space gasses produced after the flash at 9.5 min and were all identical, indicating that the product exchange reaction of the mercuric acetate traps resulted in elution profiles as shown in Chart 4A along with a chromato- was homogeneous and consisted of only a single compound, 3-ethylthiopropionate. gram obtained with a reference sample of ethanethiol (Chart Because marked effects of feeding rats diets containing 4B). A major peak was detected with the same retention time 1.5% of 3-ethylthiopropionate were observed, additional stud as a standard sample of ethanethiol. About 40% of the injected ies were conducted with the rat liver homogenate system to radioactivity was recovered as ethanethiol when the stream isolate metabolites of 3-ethylthiopropionate catabolism. The splitter system was installed so that the distribution of radio activity could be determined. The nature of the remaining 60% 1008060u40I of radioactivity is not known since it apparently did not elute from the column. Another possibility is that the efficiency of collection of ethanethiol may be low, thus accounting for in complete recovery. Nevertheless, a radioactive peak that co- chromatographed with ethanethiol was observed, indicating that ethanethiol is an intermediate of 3-ethylthiopropionate 20S catabolism and therefore of ethionine catabolism. 05"Â°fc h||l'IÃ• 1. II 1,1IlILi,i,. Additional experiments were conducted to determine if hy drogen sulfide was produced during 3-ethylthiopropionate ox idation as was observed during the oxidation of 3-methylthiopropionate (39). Gasses produced from the incubation of 3- 6040200A----B-.31,M'1 ethylthiopropionate at pH 6.5 (39) in the rat liver homogenate system were trapped in a zinc acetate solution and analyzed for hydrogen sulfide by the mÃ©thylÃ¨neblue method (34). This 1IIil. assay will detect as little as 50 nmol of hydrogen sulfide (39). 1---.h---ii40 111llljlilil, Il 1Â»!IIIIÂ«lu,. The test for the formation of mÃ©thylÃ¨neblue was negative 50 60 70 80 90 100 110 120 130 I4C indicating that hydrogen sulfide, if formed, was further catab- MASS Cm/a) olized at a rate sufficient to prevent its accumulation. Chart 3. Representative mass spectra of the gas Chromatographie effluent of the peak at 9.5 min (Chart 2). Gas chromatography was performed as described A final experiment was conducted to determine the extent of in Chart 2. Mass spectroscopy of the gas Chromatographie effluent was per oxidation of [ef/jy/-1-14C]-3-ethylthiopropionate to 14CO2in the formed as described in the text. A, compound isolated from rat liver homogenate incubation corresponding to the peak at 9.5 min in Chart 2A. B, reference 3- liver homogenate. The gasses evolved during the incubations ethylthiopropionate corresponding to the peak at 9.5 min in Chart 2ÃŸ. were first swept through a solution of mercuric acetate to

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Ã±.D. Steele and N. J. Benevenga remove [efr/y/-1-14C]ethanethiol and other volatile organic sul viving animals, 2 rats exhibited partial paralysis, and one fur compounds followed by an ethanolamine:methyl cellosolve showed evidence of moderate nasal hemorrhage. The hema- (1:2, v/v) trap and counted for radioactivity. The oxidation of tocrits of the surviving animals at the end of the experimental 10 mM [ef/iy/-1-'4C]-3-ethylthiopropionate to CO2 at pH 7.7 in period were decreased significantly as compared to control the rat liver homogenate system resulted in the recovery of rats (34 versus 38%, p < 0.01). The kidneys were enlarged 1.83 Â±0.07 (A/ = 5) junol CO2 per g wet tissue per hr. This substantially (1.31 versus 0.71 g/100 g body weight, p < was approximately twice the recovery observed when ethionine 0.001), and the spleens were very dark but not enlarged as was incubated with pyruvate (Table 1). This coupled with the was reported for rats fed 3-methylthiopropionate (36). recovery of the ethylcarbon of ethionine as the keto acid (16.38 /^mol/g/hr) when pyruvate was supplemented (Table 1) indi cates that the decarboxylation of a-keto-Y-ethiolbutyrate to 3- DISCUSSION ethylthiopropionate might be the limiting step in the transaminative pathway for ethionine catabolism. These experiments demonstrate clearly that ethionine may With the identification of 3-ethylthiopropionate and ethane- be oxidized by a transaminative pathway similar to the pathway thiol as intermediates in the oxidative catabolism of ethionine, that has been described for methionine catabolism (27, 35, 37, it was decided to feed 3-ethylthiopropionate to rats to deter 39). Based on the results reported here and of others (11, 21, 27), ethionine may be transaminated to a-keto-y-ethiolbutyrate mine if it possessed any of the toxic properties of ethionine or their biological analogs, methionine or 3-methylthiopropionate. and then apparently decarboxylated to carbon dioxide and 3- ethylthiopropionate. This is the first demonstration of 3-ethyl The effects of feeding rats for 2 weeks a 10% casein diet supplemented with 1.5% of 3-ethylthiopropionate are shown in thiopropionate formation in a mammalian system. However, Chart 5. The body weights of animals consuming the 3-ethyl studies with Escherichia coli have demonstrated the formation of 3-ethyl[35S]thiopropionate from either [35S]sulfide or [35S]- thiopropionate diet were depressed after only 1 day and re mained so for the remainder of the 2-week experimental period. ethionine (14). The complete metabolic fate of 3-ethylthiopropionate is not Food intakes were reflective of body weight changes (data not known, but carbon-sulfur bond cleavage does occur at some shown). Whereas the control animals approximately doubled their weight over the 2-week period, the animals fed 3-ethyl point yielding ethanethiol and an unknown product, possibly acrylic acid. Brown and Scholefield (5) have observed the thiopropionate did not maintain their initial weight. formation of 14CO2 from 3-ethylthio-{1-"lC]propionate in rat The effects of feeding rats 3-ethylthiopropionate were not confined to changes in body weight and food intake. The liver slices. Kidney and heart also showed significant activities, breath of the animals had the very distinctive smell of ethane- and a small degree of oxidative capacity was observed in spleen and lung preparations. Ethanethiol is apparently catab- thiol (ethyl mercaptan) after only 1 day of feeding. Gross olized further since '"CO2 was recovered from incubations with neurological changes were observed in each animal with a either [efr)y/-1-'4C]-3-ethylthiopropionate or ethionine. Recent marked lethargic condition being the most obvious. Mortality during the 2-week experimental period was 40%. Of the sur- evidence by Farber (16) on the mechanism of S-dealkylation supports our conclusions. Ethanethiol was found to be an excellent substrate for a soluble deethylating enzyme and IBO yielded acetaldehyde as a product. Natori and Tarver (28) had earlier demonstrated the formation of acetate from ethionine. Thus, the fate of the ethyl carbons of ethanethiol is likely to be oxidation to CO2 via the intermediate formation of acetaldehyde and acetate. The existence of ethanol as an additional inter mediate in the oxidation series is possible also since labeled ethanol was isolated from rats given injections of L-[efriy/-1- 14C]ethionine (28). The fate of the sulfur atom of ethionine and hence ethanethiol is not documented. However, based on our earlier observations with methionine catabolism and methanethiol oxidation (39), we suspect that the sulfur atom of ethanethiol is probably oxidized to sulfate via hydrogen sulfide, thiosulfate, and sulfite formation. The results in Table 1 and Chart 1 indicate that this trans aminative pathway for ethionine catabolism probably repre sents the major route for ethionine oxidation in vitro, since 14CO2 production from [efriy/-1-14C]ethionine can be totally suppressed by expansion of the 3-ethylthiopropionate-trapping pool. The significance of this pathway in vivo has not been 6 8 I2 I4 DAYS ON DIET investigated. The appearance of the a-keto acid of ethionine in Chart 5. Average daily body weights of rats fed a 10% casein control diet the urine of animals fed ethionine, however, suggests that this (A) and of rats fed the control diet supplemented with 1.5% of 3-ethylthiopro pathway is operative in vivo (3, 33). pionate (â€¢)Each point represents the mean of 5 rats except for animals fed the 3-ethylthiopropionate diet, where fV = 4 from Days 8 to 13 and N = 3 on Day 14. Ethionine administration to animals has resulted in a number The coefficients of variation were less than 10% for each point of biochemical alterations in addition to the development of

3940 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Mammalian Ethionine Metabolism hepatic carcinoma. Some of these observations include deple 19. Harper, A. E.. Benevenga, N. J., and Wohlhueter. R. M. Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev., 50. 428-558. tion of liver glutathione (17, 20), decreased levels of hepatic 1970. ATP (29, 43), disaggregation of liver polysomes (42), inhibition 20. Hsu, J. M., Buchanan, P. J., Anilane. J., and Anthony, W. L. Hepatic of RNA synthesis (12, 41), microsomal membrane alterations glutathione concentrations linked to ethionine toxicity in rats. Biochem. J., / 06 639-643. 1968. (24, 25), and ethylation of tRNA (30, 31). It is not known 21. Ikeda, T., Konishi, Y., and Ichihara, A. Transaminase of branched chain whether all of the changes observed after ethionine administra amino acids. XI. Leucine (methionine) transaminase of rat liver mitochondria. tion can be accounted for solely through formation of S-aden- Biochim. Biophys. Acta. 445 622-631. 1976. 22. International Committee for Standardization in Haematology. Recommen osylethionine. Studies by a number of investigators have dations for haemoglobinometry in human blood Br. J. Haematol. Â»3(Suppl). shown, for example, that ethylation of tRNA by ethionine is not 71-75, 1967. totally due to S-adenosylethionine formation since complete 23. Jeffay. H., and Alvarez, J. Liquid scintillation counting of carbon-14: Use of ethanolamine-ethylene glycol monomethyl ether-toluene. Anal. Chem.. 33 inhibition of S-adenosylethionine formation by methionine or 1- 612-615, 1961. aminocyclopentanecarboxylic acid did not completely inhibit 24. Kisilevsky, R., and Weiler, L. A microsomal membrane alteration following ethionine intoxication. Cancer Res.. 34: 3421-3427. 1974. ethylation of tRNA by ethionine (30, 31). Whether the trans- 25. Kisilevsky, R.. and Weiler. L. Microsomal membrane alterations during acute aminative pathway of ethionine metabolism reported here is ethionine toxicity and carcinogenesis. Exp. Mol. Pathol., 24: 193-200, involved in any of these metabolic changes due to ethionine 1976. 26. Mitchell, A. D., and Benevenga. N. J. Importance of sarcosine formation in administration appears worthy of further investigation. methionine methyl carbon oxidation in the rat. J. Nutr., (06. 1702-1713. 1976. 27. Mitchell. A. D., and Benevenga, N. J. The role of transamination in methionine REFERENCES oxidation in the rat. J. Nutr., 708. 67-78, 1978. 28. Natori, Y., and Tarver, H. Studies on ethionine. VII. Formation of ethanol and 1 Beliveau, G. P. A study of methionine transamination in rats. M. S. Thesis, acetate from the ethyl group of ethionine in the rat. Biochim. Biophys. Acta. University of Wisconsin, Madison, Wis., 1976. Â»07.136-138, 1965. 2. Benevenga. N. 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OCTOBER 1979 3941

Robert D. Steele and Norlin J. Benevenga

Cancer Res 1979;39:3935-3941.

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