ETHIONINE CARCINOGENICITY AND OTHER BIOLOGICAL PROPERTIES.. ACTIVATING AND DETOXIFYING METABOLISM. MECHANISM OF CARCINOGENIC ACTION David Y. Lai, Ph. D., Joseph C. Arcos, D. Sc., and Mary F. Argus, Ph. D. Drepared for the Chemical Hazard Identification Branch "Current Awareness" Program ------- Table of Contents; 5.2.1.5 Ethionine 5.2.1.5.1 Historical Background 5.2.1.5.2 Acute Biological Effects 5.2.1.5.3 Carcinogenic Activity 5.2.1.5.4 Modification of Carcinogenesis 5.2.1.5.5 Metabolism 5.2.1.5.6 Mechanism of Carcinogenic Action References ------- 545 5.2 1.5 Ethionine 5.2.1.5 1 Historical Background Ethionine is the ethyl analogue of the naturally occurring essential ammo acid, rnethionine It was first synthe- sized and studied by Dyer (1) in 1938 In her experiments on the physiological COOH COOH I I NH - CH- CH - CH -S - CH - CH NH - CH- CH - CH -S - CH Ci C* b £* j Cn C* C* j Ethionine Methionme specificity of methionine with reference to the methylthiol group, she found that ethionine could not substitute for methionine in supporting the growth of rats A few years later, similar observation was made by Harris and Kohn (2) who noted the growth inhibition of Escheri.ch.ia coli by ethionine Supple- mentation with methionine of the medium counteracted the effect Subsequent studies by other investigators have shown that ethionine is a metabolic anta- gonist of methionine, inhibiting the growth of a wide variety of microorganisms, as well as inducing biochemical and pathological injuries in various organs of higher animals (3) The carcinogenic activity of ethionine was first indicated in a study by Popper et al. (4) in 1953, tumor-like nodules were induced in. the liver when rats were maintained for a prolonged period of time on an ethionine-contain- ing diet This was confirmed by Farber (5, 6) who,' in addition, noted the in- vasiveness and metastasis of these tumors when the animals were maintained for longer periods of time on ethionine diet Subsequently, the induction of unequivocal liver carcinomas by the chronic administration of ethionine was demonstrated by other investigators (e_ _g_ , 7-9) ------- 546 Early work dealing with the toxicity and metabolism of ethionine was re- viewed by Stekol (10) and by Farber (3), and its physical and chemical proper- ties were described by Greenstein and Winitz (11) The resemblance of ethi- onine to methionine in chemical reactivity and cellular metabolism renders ethionine an unusual, important tool in the study of the possible mechanism of liver tumongenesis For some time, ethionine was regarded only as a labor- atory curiosity However, Schlenk (12) reported in 1957 that S-adenosylethi- onine, the sulfur activation product of ethionine, was produced in yeast cells exposed to ethyl mercaptan Evidence for the biosynthesis of ethionine in sev- eral strains of bacteria was presented a few years later (13) and of particular importance was the fact that some of these ethionme-producmg bacteria are present in the normaffilora of the mammalian intestinal tract Because of its /** carcinogenic activity as well as its synergistic effects with other carcinogens (see Section 521 5.4) the natural occurrence of ethionine may have a speci- al etiological significance 521 52 Acute Biological Effects. The biological effects of ethionine have been extensively studied in microorganisms and higher animals In vir- tually every organism or species studied, ethionine causes inhibition of growth or weight loss (3, 14) In higher animals, ethionine brings about various physi- ological and pathological effects, including acute effects in the liver, pancre- as, kidney and other organs (Table CXI). Many of these effects can be re- s* y / versed by methionine treatment, indicating that the biological effects of ethi- onine are due to antagonism with methionine ------- Table CXI Acute Biological Effects of Ethionine p. 1 of 2 pp. Organ or tissue Species Effects References Liver Rat Pancreas Mouse Guinea pig Dog, cat, monkey, chick Rabbit Rat Guinea Pig Mouse Rabbit Fatty degeneration of the liver (steatosis) in females. Decrease in the extent of liver regeneration after partial hepatectomy. Steatosis. Steatosis. \ Steatosis. Steatosis. Hypercholesteremia, jaundice, and changes in bile capillaries. Acute acinar necrosis. Pancreatic degeneration. Varying degree of pancreatic acinar destruction. 15, 41-43 44 45 46-48 49, 50 51 45 44 49, 52 ------- Table CXI continued p. 2 of 2 pp. Kidney Testis Digestive tract Embryo Dog Cat, monkey Rat, Cat, dog, monkey Rat Rat Dog, rabbit Rat Rabbit, dog Chick Pancreatitis. Decrease of external pancreatic secretion. Fatty change in tubules, Necrosis of distal portion of proximal convoluted tubule. Fatty change in tubules; Necrosis of distal portion of proximal convoluted tubule. Progressive degeneration of tubular cells, beginning with spermatozoa. j Hemorrhage. Adrenal cortical hyperplasia. Degeneration of chief cells of gastric mucosa, and cells in salivary glands and duodenum. Gastrointestinal hemorrhage. Growth inhibition induction of fatty liver and edema. 49, 53 46-48 15, 54 46-48 55-57 15 49 58 49 59 Patterned after E. Farber Adv. Cancer Res. T_> 383 (1963) . ------- 547 The major acute consequence of the administration of ethionine to ani- mals is the induction of fatty degeneration of the liver (steatosis) In rats only the female but not the male develop this syndrome following ethionine adminis- tration (15, 16) This was suggested to be the result of hormone-dependent specific metabolic differences (16, 17). It has been shown that parenteral ad- ministration of ethionine to female rats and other animals inhibits the synthe- sis of protein (18-20) and RNA (21-24) in the liver, and causes disaggregation of the polyribosomes (20, 25) Several investigators have suggested that the basic biochemical defect in steatosis may be a block in the secretion of tn- glycerides by the liver, and that this block may be due to the inhibition of the hepatic synthesis of the protein moieties of serum lipoprotein (26, 27) Recent work has shown that ethionine impairs protein synthesis by reducing the rate of chain initiation (28-30) However, it is generally considered that the acute ef- fects of ethionine are not caused by its direct action on the protein synthesiz- ing system, but rather indirectly by interference with ATP metabolism through the trapping of adenine (31). There is indeed a drastic decrease of ATP (32) and an accumulation of S-adenosylethionine (33) in the livers of female rats, following ethionine administration The observation that the administration of ATP or adenine counteracts the induction of steatosis or the inhibition of ami- no acid incorporation into liver microsomes, lends further support to this hy- pothesis (32, 34) Other biochemical mechanisms of possible pathogenic significance re- garding cell injury by ethionine include (a) synthesis of abnormal proteins by ------- 548 ethiomne incorporation substituting for methionine, (b) production of ethylated instead of methylated cellular macromolecules and constituents, and (c) for- mation of toxic metabolic intermediates during ethiomne metabolism These processes are discussed in some detail in Section 5.2.1.5 5. The acute toxicity of ethiomne has been tested in several species of lab- oratory animals. Farber _et al_ (1 5) noted that death of fasted female rats (100-200 gm) occurred within 30-50 hrs of the first dose when they were ad- ministered 200 mg DL-ethionine in four doses spaced at 2j hrs The acute, 7-day LD of D-ethionine in Swiss mice dosed intraperitoneally is 185 mg/kg (35) In guinea pigs, 1, 000 mg/kg DL-ethionine in a single dose was lethal to males, while females tolerated slightly higher doses (1,000-2,000 mg/kg) (36) Death occurred within 8 hrs. after administration Simultaneous admimstra- i tion of methionine could, however, prevent lethality. When pregnant rats were fed diets containing ethiomne, fetal resorption and induction of various anomalies were seen (37) Ethionine is transported across the placenta of rats and mice and is incorporated into proteins of the fetus (38-40) 5.2 1.5 3 Carcinogenic Activity. Although the mutagemcity of ethiomne has not been established in the Salmonella test system with or without liver microsomal activation (60), its carcinogenicity has.been clearly demonstrated. Popper and his associates first noted the development of tumor-like nodules in the liver in 8 of 12 rats fed a diet containing 0. 5% ethiomne for 51 days (4) Despite the histological features suggestive of abnormal growth and anaplasia, ------- 549 clear-cut evidence of malignancy was obtained only later by Farber, who found 7 of 12 tumors showing invasion and metastasis (5). These unequivocal hepa- tomas were induced by the administration of semisynthetic diets containing 0.25% DL-ethionine for periods of 8 to 10 months Similar findings were made ^*-~ "i -j by other investigators (Table CXII). The incidences of hepatic tumors show a L. X{( dose-response relationship and increase with the duration of administration (3). The ethionine-induced tumors are usually multiple, consisting of nod- ules measuring from 1 to 8 cm in diameter and are almost exclusively hepa- tocellular in nature. Metastatic growth most frequently involves the lung, the pancreas and the omentum Many of these hepatomas are transplantable (6l, 62). Prior to the appearance of the malignant tumors the following sequence of events occurs (a) oval cells appear after one to three weeks, (b) bile duct proliferation takes place within 2 months, and (c) nodular hyperplasia is noted after 6 to 12 weeks. The detailed morphological, histochemical and biochem- ical changes in the livers of rats induced by dietary administration of ethionine r have been described (3, 63). Studies on the carcinogenic activity of ethionine have been carried out al- most exclusively in rats with the exception of the reports on the hamster by Hancock (64), and by Terracini and Delia Porta (65). They found that chronic administration of ethionine to Syrian golden hamsters failed to produce ductu- lar proliferation, nodular hyperplasia or liver carcinoma This observation is consistent with other findings that due to basic metabolic differences the hamster reacts differently from the rat to many hepatic carcinogens (66, 67) ------- Table CXH Hepatocarcmogenicity of Ethtorune p. 1 of 3 pp. Species Strain and sex Rat Wistar, M & F Wistar, M & F Wistar, M & F Wistar, M & F Wistar, M & F Wistar, M & F Wistar, M & F Wistar, M Wistar, M Wistar, M Dietary level and duration of treatment 0. 25% for 8-10 mo. 0. 25% for 8 mo. then normal diet for 4 mo. 0. 25% for 10 mo. 0. 5% for 8-11 mo. 0. 25% for 5 mo. followed by normal diet for 6 mo. 0. 25% for 5 mo. 0. 25% for 7j mo. 0. 1% for 34 wk. 0. 25% for 5 mo. 0. 25% for 20 wk. followed a Tumor incidence 12/14 8/10 8/11 10/20 \ 9/12 \ 30/35 13/13 3/12 6/11 9/12 References 5 77 63 9 83 3 3 71 87 75 by normal diet for 4 wk. ------- Table CXII continued p. 2 of 3 pp. Wistar, M Wistar, M Wistar, M Fischer, M & F Fischer, F Fischer, M Sprague-Dawley, M & F Sprague-Dawley, M Sprague-Dawley, M Sprague-Dawley/ — Osborne-Mendel, M & F CFN, F 0.25% in semisynthetic diet for 34 wk. then chow fo r 1 8 wk. 0.2% for 9 mo then normal diet for 6 mo. 0.25% for 31 wk, 0. 25% for 7j mo. 0. 25% for 9 mo. 0. 25% for 24 wk. 0.25% for 38 wk. 0. 05% in choline-devoid 0.25% for 42 wk. 0. 25% for 8 mo. 0. 25% for 8-9 mo. 0. 3% for 8 mo. 30/30 11/16 68 19% 17/18 10/31 100% 28/38 13/16 8/12 12/15 10/18 12/15 78 3 88 89 73 70 76 90 8 72 ------- Table CXII continued p. 3 of 3 pp. Holtzman, M Holtzman, M Holtzman, F Mouse Not specified Hamster Syrian golden, M & F Syrian golden, M & F 0. 25% for 5 mo. 0. 25% for ?i mo. 0. 25% for 7j mo. Not specified 0. 2% for 150 days 0. 2% in drinking water for 34 wk. 0/32 6/10 2/8 b Liver tumor 0/35 0/18 3 3 3 64 64 65 No. of rats with hepatoma/effective group Incidence not reported ------- 550 Ethionine has been claimed to induce hepatomas in mice (64), but this obser- vation has not been confirmed In addition to species differences, strain dif- ferences have an important influence, Farber and Ragland (cited in ref. 3) noted a significantly lower susceptibility of Holtzman strain rats to the induction of liver tumors by ethionine than other strains (see Table CXII ). The specificity of the molecular structure of ethionine to induce tumors has been studied by Argus et al (68) They found that S-ethyl-L-cysteme, a lower homolog of ethionine, is not carcinogenic to male Wistar rats at approx- imately three times the dose at which ethionine produces 100% liver tumor in- cidence The result was interpreted that the 4-carbon backbone common to the ethionine and methionine molecules is necessary for the hepatocarcmogen- ic activity of ethionine Although S-propylhomocysteine and S-JSoamylhomo- cysteine are also activated _in vitro to their respective S-adenosyl derivatives and induce in rats similar initial histological changes asaie induced by a com- parable dose of ethionine (69), no other S-alkyl-homocysteine except ethionine has yet been found to induce hepatomas in any animal species. 'This indicates that the metabolic lability of the ethyl group of ethionine critically determines the carcinogenic activity of this ammo acid analogue 5.2 1 5.4 Modification of Carcmogenesis. Shinozuka £t al (70) have shown that a cholme-deficient diet modifies the resp'onse of rat liver to DL-ethio- nine and leads to early and enhanced induction of hepatocellular carcinoma. A diet supplemented with 0 5% lithocholic acid, an important metabolite of chol- esterol, was also found to promote the development of hepatic hyperplastic ------- 551 nodules and hepatomas induced by DL-ethionme in the rat (71). On the other hand, a number of compounds have been found to have inhibitory effect on liv- er tumongenesis due to ethionine. For instance, Brada and Bulba (72) re- ported that oral administration of 1, 1 0-phenanthrohne (0 05%), an iron chelat- ing agent, significantly inhibits the induction of carcinomas in livers of ethio- nine-mgesting rats, Male or female rats fed a diet containing orotic acid eith- er 3 weeks before or during the feeding of ethionine develop fewer liver tumors than rats fed ethionine alone (73). Addition of 0. 25% copper acetate totally in- hibits the induction of hepatomas in rats receiving diets containing 0. 25% ethio- nine foi 24 weeks (74, 75). The inhibitory effect of o(-naphthylisothiocyanate (ANI) on liver tumor induction by ethionine has also been reported (76) The mechanisms of the inhibitory effect of these agents is unknown However, the notion that ethionine may induce liver cancer by interference with the metab- olism of methionine is supported by the finding that addition of 0 6 or 0 8% DL-methionine completely prevents the induction of liver tumors in rats by ethionine (77) Syncarcinogenic effect between ethionine, on one hand, and 4-dimethyl- aminoazobenzene (78, 79) or 2-acetylaminofluorene (80), on the other hand, has been shown Denda and coworkers (81, 82) reported that pancreatic tumor- igenesis by azaserine or 4-hydroxyaminoquinoline-l-oxide in the rat is en- hanced by giving 0 5% ethionine in the diet Interestingly, commercial trypan blue and ethionine are mutually antagonistic in that trypan blue suppresses the induction of ethionine-induced hepatocellular carcinomas and ethionine ------- 552 suppresses the induction of trypan-blue-mduced reticulum cell sarcomas (83). The mechanism(s) of these interactions is presently unknown Unlike the in- hibition by 3-methylcholanthrene of liver tumor induction by certain other he- patic carcinogens (84-86), however, 3-methylcholanthrene has no effect on the development of hepatocarcinoma induced by ethionine, this suggests (87) that microsomal metabolism of ethionine may not be involved in ethionine carcin- ogenes is 52155 Metabolism The metabolism of ethionine has been the sub- ject of considerable study since the early discovery of ethionine hepatotoxicity and carcinogenicity The information available strongly indicates that the bio- chemical systems that metabolize ethionine are virtually the same as those - that metabolize methionine, although very large differences exist between the rates at which the ' enzymes react with ethionine and methionine It is the rel- ative activities of various enzymes upon these two substrates or their metab- olites that may be the factors that determine the biological effects of ethionine Early studies of ethionine metabolism revealed that following i p admin- istration, ethionine becomes widely distributed in the tissues of the rat (91, 92) However, the distribution is not uniform In all organs studied, the maximum 14 level of acid-soluble radioactivity following the administration of ethyl- C- -ethionine was observed at or after 8 hrs Levine and Tarver (91) reported that kidney contains the highest concentration of ethionine metabolites, followed by liver, small intestine, plasma, and spleen. More recently Brada et al, (93) showed that the highest level of acid*-soluble ladioactivity following oral ------- 553 (the) administration of radioactive ethionine was yirver, the only carcmogenicity target organ of ethionine. The two groups of investigators, however, do agree in their findings of longer persistence of ethionine in the kidneys than in oth- er organs. The metabolism of ethionine in higher animals proceeds following the rlQ i jb O four basic metabolic pathways (i_ through rv) shown in Fig 28 Oxidation to Carbon Dioxide (i) Several studies have revealed that the relative rate of oxidation of ethionine to CO is much slower as compared to c* methionine Farber and Magee (94) reported that the amount of respiratory 1 4 CO released by rats after application of ethyl- 1- C-ethionine was less than 14 7% of that from rats given an equal amount of C-methyl-methionine Simi- lar observation was made by Levine and Tarver (91) who demonstrated that 1 4 only about 3% of the administered dose of ethyl-1- C-ethionine was recovered 14 as CO£ after 24 hrs This finding was confirmed by Stekol _e_t al_ (95) who, in addition, noted that male rats have a greater capacity to oxidize ethionine than do female rats. Recent work by Brada jet al (93) showed that about 6% of an 14 orally given or i. p. injected dose of ethyl-1- C-ethionine was recovered as 14 CO2 after 24 hrs The enzymatic mechanism for the oxidation of ethionine to CO is not well understood Investigations by Steele and Benevenga (96, 97) suggest that ethionine may be catabolized by a transaminative route similar to the pathway f~Z,Q vr "*"~ postulated for methionine catabolism (98). As illustrated in Fig 29, ethionine y ^^ is first transaminated to o(-keto-0 -ethylthiobuty rate andV'^^decarboxylated to ------- N-ocetylethionme sulfoxide Ethionme sulfoxide Oxidation (11) Sulfur activation ATP- Corboxyl activation S-odenosylethionme Transethylation Ethyl derivatives of macromolecules Figure 28 ------- LEGEND TO FIGURE 28 Fig. 28. Basic metabolic pathways of ethionine ------- COOH 1 H2N-CH CH2 1 - CH2 1 | C2H5 Ethionme COOH OH i i i i r\ r* — r\ ""U ~^ ^~U 1 C02 I Transamtnotion CH2 J CH2 S X CH2 CH2 / \ i i ' IS S Keto Ammo | | acid acid C2H5 C2H5 a-keto- 3-ethyl- /-ethyl thio- -thiobutyrate propionate • SH-C2H5 \ Ethanethiol H2S CH3-C-H — Acetaldehyde •M ze CH3-C-Oe- Acetate -C02 H2C=CH-COOH Acrylic acid Figure 29 ------- LEGEND TO FIGURE 29 Fig. 29. Transaminative pathway of ethionine catabolism. ------- 554 CO_ and 3-ethylthiopropionate, which is further catabolized to ethanethiol and probably acrylic acid. Oxidation of ethanethiol yields CO_ via the intermedi- L* ate formation of acetaldehyde and acetate The sulfur atom of ethanethiol is suspected to be oxidized to sulfate via hydrogen sulfide In view of the toxic- ity of 3-ethylthiopropionate produced in this pathway, it was suggested that (9?) this oxidative pathway may be intimately involved in the pathogenic effects of ethionine. Oxidation to ethxonine sulfoxide with subsequent acetylation (u) In con- trast to the minor route of ethionine oxidation to carbon dioxide, 70% or more 1 4 of the'admmistered C is excreted in the urine of rats injected with ethyl-1- 14 C-ethionme within 24 hrs. (91-93). Four main components, which account for about 90% of the radioactivity in the urine have been identified as N-acetyl- ethionine sulfoxide, ethionine sulfoxt.de, free ethionine and S-adenosylethionine (93, 99, 100) These compounds were also demonstrated to be major metab- olites in various organs including liver, kidney, small intestine, and blood of rats after oral administration of ethionine (93). Data obtained by several in- vestigators support the idea that the major pathway for the metabolism of ethio- nine in the rat is ethionine ^ ethionine sulfoxide ^ N-acetylethionine sulfoxide (93, 100) The biological and toxic effects of these ethionine metab- olites are not clear Inasmuch as acetylation of amines and oxidation of sul- fur compounds to the sulfoxides may be regarded as pathways of detoxification, it is possible that these metabolic conversions are mechanisms for the detox- ification of ethionine (100) Although ethionine sulfoxide has been shown to ------- 555 produce steatosis after i. p injection to lats, the activity is believed to depend on its reduction to ethionme (101, 102) Sulfur Activation to S-Adenosylethionine (111). The biological activity of methionine is largely determined by its activation to S-adenosylmethionine (SAM) and subsequent transfer of the methyl group from SAM to various accept- ors. In analogy with SAM, S-adenosylethionine (SAE) is formed in vivo in rats either fed a diet containing ethionine or injected with ethionine (33, 93, 103-107) The studies of various investigators (108, 109) have established that ethionine reacts with ATP to form SAE in the liver via the same enzyme (ATP L-methio- nine S-adenosyltransferase) which catalyzes the formation of SAM The trans- fer of the ethyl group from SAE by transethylation to various naturally occur- ring compounds which normally accept methyl groups^ has also been established (94, 95, 110, 111) Stekol and Weiss (110) hypothesized that one or more of the^ ethyl analogues of such compounds may be responsible for the pathogenesis of ethionine-induced hepatic lesions This subject will be further discussed in Section 5. 2. 1. 5 6. While the respective S-adenosyl derivatives of ethionine and methionme are formed by the methionine-activating enzyme to approximately the same ex- tent, the extent of transfer of the ethyl group of SAE to various tissue accept- ors is much smaller than that of methyl group transfer from SAM (95, 106) As a consequence of this imbalance between the rates of formation and utiliza- tion of SAE, accumulation of SAE occurs in the liver, which is considered as an "ATP-trapping action", accounting for the rapid fall in the concentration of ------- 556 ATP in the liver of rats administered ethionine (19, 31-33, 112) Previous studies concerning the hepatotoxic effect of ethionine have revealed that fol- lowing the depletion of hepatic ATP there is a striking inhibition of protein (19, 34, 113) and nucleic acid (21-23) synthesis. These metabolic changes were sug- gested to be intimately related to the induction of steatosis and other tissue le- sions by ethionine (26, 27, 105, 114). Carboxyl Activation (iv). Incorporation of ethionine into proteins has been demonstrated to occur iri vivo in rat (40, 91, 92, 115, 116), mouse (117), cat (118), protozoa (119) and bacteria (120), as well as in vitro in Ehrhch asci- tes carcinoma cells (121). The first indication of carboxyl activation of ethio- nine was provided by Berg (122) who showed that an enzyme preparation from yeast which activated methionine to methionme adenylate was also able to generate ethionine adenylate from ethionine and ATP. Glenn (123) studied the carboxyl activation of L-methionme and L-ethionine in the rat and found the ex- istence of a common enzyme toward the two substrates in rat liver However, the affinity of the enzyme for ethionine is much lower than that for methionine Further evidence in support of carboxyl activation of ethionine was offered by Villa-Trevino and Farber (34) They found that t-RNA isolated from the liver of rat li to 5 hrs after the administration of ethyl-1- C-ethionine was radio- active and the radioactivity could be largely removed by incubation of the t-RNA at pH 10, a procedure which is known to remove the ammo acid bound in ester linkage to t-RNA after activation (124) 5.2.1 56 Mechanism of Carcinogenic Action While the carcmogen- icity per se of ethionine has been clearly established, little is known about the ------- 557 mechanism underlying its carcinogenic action The reactions which may be significant in carcinogenesis are the production of abnormal ethylated ana- logues of naturally occurring methyl-containing compounds. There is indeed extensive evidence showing the incorporation of ethyl group from the ethyl-1- 14 - C-ethionme into proteins (40, 91, 92, 115-118, 125), nucleic acids (94, 125, 126) and other cell constituents such as choline and creatine (110), and histidine and carnosine (111), to give ethylated derivatives , ' A For some time it was held that the incorporation of ethionine, in place of methionine, into protein may be an important biological aspect related to carcinogenesis (127, 128) Conceivably, the substitution of ethionine for methio- nine could lead to the production of abnormal proteins which could, in turn, trig- ger the neoplastic transformation. However, the idea is inconsistent with the evidence obtained from ethionine incorporation into protein in various tissues Both kidney and intestinal mucosa incorporate ethionine more actively than liver (91) Yet, liver is the only target organ of ethionine carcinogenesis in the rat It was also believed earlier that the effects of ethionine on the production of energy in the cell is a factor in its carcinogenic activity (3) However, later findings indicated that alterations of the mitochondrial ATP synthesizing sys- tem as well as damage to the availability of ATP caused by ethionine, ob- served only in females, are related only to the induction of steatosis but not to its hepatocarcinogenic action, to which both male and female rats are suscep- tible (68) ------- 558 The presently held concept is that introduction of the ethyl group of ethio- nine in lieu of the methyl group of methionine into DNA and RNA is the key bio- chemical event, since the interaction of nucleic acids with chemical carcinogens is generally regarded as the initiating event in carcmogenesis (126, 129-131) Unlike most carcinogens, however, ethionine does not react extensively with DNA (132-134). The radioactivity which remains associated with the DNA iso- 14 lated from the liver of rats following injection of ethyl-1- C-ethionine repre- sents only one ethyl group for every 20x10 nucleotides (134) This is one or two orders of magnitude less than the labelling observed with polycyclic hydro- carbons, whose mode of carcinogenic action is generally believed to involve binding to DNA (135) Farber and coworkers (125) showed that following mjec- 14 tion of ethyl-1- C-ethionine to the rat, all liver RNA fractions were labelled, i with t-RNA being'the most highly labelled. Similar results were obtained by Natori (136) and by Ortwerth and Novelli (132) who found, in a chromatographic study, that t-RNA is the only fraction labelled to a significant extent The ob- servation is in good agreement with the fact that l-RNA is also the most active methyl group acceptor (137). Several other hepatic carcinogens, 4-dimethylaminoazobenzene, benzo[a ]- pyrene and 2-acetylaminofluorene, are known to bind in vivo preferentially to the t-RNA fraction of the target tissues (132, 138-14Q) Wemstem et al (140) suggested that cellular RNA, particularly t-RNA, may be the critical target during the chemical induction of cancer Qualitative and quantitative changes in the t-RNA population during tumorigenesis have been observed (137, 141, 142) ------- 559 _ It was hypothesized that alteration of minor nucleotides in the maturation of t-RNA could be a key event in the carcinogenic process (142) The evidence which supports the concept that ethylation of t-RNA may be involved in carcinogenesis by ethionine consists of the following (a) liver t-RNA becomes labelled to a greater extent than DNA or protein following in- 14 jection of ethyl-1- C-ethionme in rats (94, 126, 132, 136), (b) significant eth- ylation of t-RNA occurs only in the liver, the only organ in which tumors devel- op in rats administered ethionine (3, 94, 125, 126), and (c) supplementation of the diet with methionine, which inhibits ethionine carcinogenesis, also marked- ly decreases ethylation of liver t-RNA (77, 94, 125, 126) Most of the ethylation of t-RNA that occurs in the livers of rats treated with ethionine is mediated by the action of t-RNA methylases acting on S-adeno- 2 sylethionine (132, 143) The ethylated nucleosides present in t-RNA are: N -ethyl, 22 22 7-ethyl, N N -diethyl-guanine, N -ethyl-N -methyl-guanine and two ethylated pyrimidines (143, 144) The specificity and functional effects of this modifica- tion were first demonstrated by Axel e_t al. (1 38) who found that chronic feeding of ethionine to rats results in the loss of two leucine-t-RNA species This led them to hypothesize that the loss of a given t-RNA could prevent the transla- tion of messenger RNAs which are codon-specific for the t-RNA, and there- by block the synthesis of one or more proteins required for cell regulation Sharma et al. (145) have presented evidence that lysine-t-RNA is a specific target of alkylation by ethionine Ortwerth and Novelli (132) have also observed preferential ethylation of a population of liver t-RNA following the administration ------- 560 of small amounts of radioactive ethionine. However, the metabolic sequelae (on) and the effects of these ethylated t-RNAs j^ethionine tumongenesis are ob- scure at present, because of our ignorance of the function of the naturally oc- curring methyl groups in the t-RNAs It is possible that the ethylation of com- ponents of t-RNA which are normally methylated might alter the chemical spe- cificity and therefore their activity in protein synthesis and the patterns of gene expression Although the presently available data indicate that only small amounts of DNA are ethylated by ethionine, the possibility that alkylation of DNA may play a crucial role in carcinogenesis by ethionine cannot be totally excluded Fare (74) and Kamamoto _e_t al (75) reported the protective action of cupric acetate against the hepatocarcinogenes is by ethionine This is consistent with the find- t ing that ethylation of rat liver DNA by ethionine is suppressed by dietary cop- per (146) Similar to other hepatic carcinogens known to interact with DNA in _ s~" vivo, ethionine also induces repair replication of liver DNA in the rat (147) There is, moreover, evidence that a small part of the ethylation of t-RNA af- ter treatment with ethionine does not proceed via the action of t-RNA methyl- ases but by direct action of a metabolic alkylating (ethylating) intermediate (132, 143) This is also the probable mechanism by which 7-ethylguanine is formed in DNA, since 7-methylguanine is not a normal component of rat liver DNA and and no DNA methylase has yet been described (143) Therefore, it is still un- clear whether the carcinogenicity of ethionine is related to the alkylation of t-RNA via the natural enzymatic alkyl-transfer pathway or to the ethylation of ------- 561 DNA and possibly t-RNA, via a chemically reactive alkylating metabolic inter- mediate Another normal cellular process, DNA methylation in the 5-position of Cbyj cytosine has been shown to be alteredjfethionine. Cox and Irving (148) reported that S-adenosylethionine, formed in vivo from ethionine, inhibits the enzymatic methylation of DNA via S-adenosylmethionine, resulting in the production of methyl-deficient DNA in regenerating rat liver Although the exact function of the methyl group is unknown, it has been proposed that methylation of mam- malian DNA may play a role in differentiation and, thus, the aberrant process may lead to tumorigenesis (149). In an interesting study of the effects of ethio- nine on the enzymatic methylation of various DNA sequence classes in P815 mastocytoma cells, Boehm and Drahovsky (150) noted that a low dose of ethio- nine inhibits the methylation of inverted repetitive sequences (type ABC.. CBA) to a higher extent than the methylation of other sequence classes.. On the basis of the suggested regulatory role of inverted repetitive sequences in mam- malian genome (151, 152), these investigators proposed the hypothesis that the change in enzymatic methylation of these sequences caused by ethionine may be related to the ethionine-induced re-expression of embryonic genes (153). Evidence is rapidly accumulating that nuclear proteins are involved in the regulation of gene expression (_e £, 154) Modification of nuclear proteins by carcinogenic agents is expected to change their interaction with DNA, which in turn could jeopardize the fidelity of gene expression. Methylation of argin- ine and lysine residues, one of the pos tsynthetic chemical modifications which ------- 562 histories normally undergo, has been repeatedly reported with S-adenosyl- methionine as the methyl donor (128, 154-156). Ethylation of histones and oth- 1 4 er nuclear protein fractions has also been shown to occur with ethyl-1- C- -ethionme (125, 128, 157) It has been proposed that interference in the normal process of histone methylation may be related to ethionine carcinogenesis (125, 157, 158) Whether the ethylation of nuclear proteins, presumably by replace- ment of some methyl groups, would have any influence on gene expression would be an interesting area of investigations. Note added after completion of Section. 5215 P J Talmud and D Lewis [Heredity, 31, 139 (1973)],reported that ethionine is strongly mutagemc in the fungus Coprirms lagopus This positive effect has not been confirmed, however, in other mutagenicity assay systems Attempts to test ethionine for mutagenicity in two E_. coli strains (C-600 and A) gave negative results (Talmud and Lewis, loc cit ) Ethionine at doses of 200 and 1000 mg/kg i was also negative in the dominant lethal mutagenicity assay system, m the mouse [Epstein, S S. Arnold, E. , Andrea, J. , Bass, W. , and Bishop, Y , Toxicol. Appl. Pharmacol , 23, 288 (1972)] ------- 563 REFERENCES TO SECTION 5. 2. 1 5 1 Dyer, H. M. J. Biol Chem 124, 519(1938). 2 Harris, J.S., and Kohn, H.I. J. Pharmacol Exp. Therap 73, 383 (1941). 3 Farber, E. Adv Cancer Res. 7, 383(1963). 4. Popper, H , de la Huerga, J. , and Yesiruck, C. Science 118, 80 (1953) 5. Farber, E. A.M. A. Arch Pathol. 62, 445 (1956). 6. Farber, E. Cancer Res. 1_6_, 142 (1956) 7 Gelboin, H. V , Miller, J.A., and Miller, E. C. Cancer Res. 1,8, 608 (1958) 8 Sidransky, H. , Clark, S. , and Baba, T. J Nat. Cancer Ins t 30, 999 (1963) 9. Wachstein, M. , and Mies el, E. J. Histochem. Cytochem 7, 189(1959) 10 Stekol, J.A. Adv Enzymol 25_, 369 (1963) 11. Greenstein, J. P. , and Wmitz, M. "Chemis try of Ammo Acids" Vol. 3, Wiley, New York, 1961 12. Schlenk, F. Arch Biochem. Biophys. 69, 67(1957). 13 Fisher, J F. , and Mallette, M.F. J. Gen. Physiol. 45, 1 (1961). 14 Yamada, M , and Takahashi, J. Brit Poult. SQL 18, 557 (1977) 15. Farber, E. , Simpson, M. V. , and Tarver, H ' J. Biol Chem. 192, 91 (1950). 16 Farber, E , and Segaloff, A J. Biol Chem. 216, 471 (1955). 17 Glaser, G , and Mager, J Israel J Med Sci 8, 1754(1972). ------- 564 18 Simpson, M. V. , Farber, E. , and Tarver, H. J. Biol. Chem 182. 81 (1950). 19. Villa-Trevino, S , Shall, K. H. , and Farber, E. J. Biol Chem 238, 1757 (1963) 20. Sidransky, H , Sarma, D.S.R., and Verney, E. Exp Mol Pathol J_5, 320 (1971) 21. Villa-Trevino, S. , Shall, K. H., and Farber, E. J. Bioi Chem. 241, 4670 (1966) 22 Farber, J L. , Shinozuka, H , Serroni, A., and Farmer, R Lab Invest. 31_, 465 (1974). 23 Swann, P. F. , Peacock, A C , and Bunting, S. Biochem J 150, 335 (1975) 24. Ashbourne, T F. , Schultz, G A , Forrester, P I. , and Hancock, R L. Physiol Chem. Phys 8, 303 (1976) 25. Villa-Trevino, S , Farber, E , Staehelm, T. , Wettstem, F O. , and Noll, H. J. Biol. Chem 239. 3826 (1964) 26 Robinson, D C , and Harris, P.M. Biochem. J 80, 361 (1961) 27 Lombard!, B , and Recknagel, R.O. Amer J. Pathol 40, 571 (1962). 28 Kisilevsky, R Biochim Biophys Acta 272. 463 (1972) 29 Murty, C N , Verney, E , and Sidransky, H.' Exp Mol. Pathol 27, 392 (1977). 30. Yavich, M. P. Biochemistry 36, 950(1971) 31 Stekol, J A , Anderson, E I , Hsu, P T., and Weiss, S Abstracts 127th Meeting Amer Chem Soc , Cincinnati, p 4c, 1955 ------- 565 32. Shall, K. H J. Biol. Chem 237, PC 1734 (1962) 33. Shall, K. H. , McConomy, J. , Vogt, M , Castillo, A , and Farber, E J. Biol. Chem. 241, 5060(1966). 34. Villa-Trevmo, S , and Farber, E. Biochim. Biophys Acta 6l, 649 (1962). 35. Friedman, M.A., Berry, D. E. , and Elzay, R. P. Cancer Letters 3, 71 (1977). 36 Sauer, F , and Sarkar, N. K. Biochim Biophys Acta 148, 579 (1967). 37. Lee, C. M., Jr., Wiseman, J.T., Kaplan, S.A., and Warkany, J. 'A.M. A. Arch Pathol. 59., 232 (1955) 38. Hansson, E , and Gazo, T. Expenentia 17, 501 (1961) 39. Proffit, W.R., and Edwards, L. E. J. Exp Zool 151. 53(1962). 40. Schaltz, P.W. , Graves, J. , and Schaltz, R. L. Arch Biochem Biophys. 104, 387 (1964). 41 Jensen, D , Chaikoff, I L , and Tarver, H. J. Biol Chem 192, 395 (1951) 42. Gershbem, L L. Amer J. Physiol 195, 670 (1958). 43. Gershbem, L L. J. Nat Cancer Inst. 35_, 591 (1965). 44. Wachstein, M. , and Meisel, E. Proc. Soc. Exp. Biol. Med. 82, 70 (1953). 45 Rice, C. E. , Boalanger, P., Plammer, P J. G. , and Annaa, E. Can J Med Sci _3J_, 343 (1953). 46 DeAlmeida, A. L. , and Grossman, M I. Gasfcroenterology 20, 554 (1952) ------- 566 47 Lin, T.M. , and Grossman, M. L Amer J. Physiol. 176, 377(1954). 48. Kroboth, F. J. , Jr., and Hallenbeck, G.A. Proc Soc. Exp Biol Med 8JJ, 145 (1955). / 49. Wang, C. , Strauss, L. , Paronetto, F. , and Aldersberg, D. A. M. A. Arch Pathol 6_5, 286 (1958). 50. Eliakin, M , Eisner, M , and Ungar, H. Bull Res. Council Israel E7. 189 (1958). 51. Farber, E. , and Popper, H. Proc Soc Exp. Biol. Med. 74, 838 (1950). s— ^- "~ 52. Plumrner, P. J G , and Boulanger, P. Can J Comp. Med 18-,-237 (1954) 53. Nagulescu, P., Harper, H. A., Crane, J. T. , and Goldman, L Amer. i ~"~-~——— J. Sarg. 102. 196(1961). 54. Wachstein, M , and Me is el, E. Proc. Soc Exp. Biol. Med. 77, 569 (1951). 55. Alvizoun, M , and Warren, S. A.M. A. Arch Pathol 57, 130(1954). 56. Kaufman, N , Klavms, J. V. , and Kmney, T.D Amer. J Pathol. 32, 105 (1956). 57. Goldberg, G. M. , Pfau, A , and Ungar, H. Amer J Pathol 35, ^49 (1959) 58. Lormg, W. E , and Hartley, L. J. Amer J. Pathol 31, 521 (1955). 59. Rizzoli, C , Dessi, P., and Cestari, A. Ricerca Sci. 29, 2405 (1959) 60. McCann, J , Choi, E , Yamasaki, E , and Ames, B. N. Proc Nat. Acad Sci (US) 72, 5135 (1975). ------- 567 61. Pilot, H. C. Cancer Res. 20, 1262(1960). 62. Potter, V. R. , Pitot, H. C. , Ono, T. , and Morris, H. P. Cancer Res. 20_, 1255 (I960) 63. Dunn, W. L. J. Pathol Bactenol. 89. 513(1965) 64. Hancock, R L. Physiol Chem Phys. 4, 573 (1972). -; '. 65. Terracmi, B. , and Delia Porta, G. A.M. A. Arch PathoL 71, £66 (1961). 66 Homburger, F. Progr. Exp Tumor Res. l6, 152 (1972). 67 Brada, Z. , Bulba, S. , and Altman, N. H. Proc Amer. Assoc. Can- cer Res. 1_8, 239 (1977). 68. Argus, M.F., White, L E. , Bryant, G.M., Arcos, J. C. , and Hoch- -Ligeti, C Z Krebsforsch. 75, 201 (1971). 69 Sfcekol, J.A. In "Transmethylation and Methionine Biosynthesis" (S.K Shapiro, and F. Schlenk, eds ) Univ Chicago Press, Chicago, 1965, p. 231. 70. Shinozuka, H , L-ombardi, B. , Sell, S. , and lammanno, R.M J. Natl Cancer Inst 6j_, 813 (1978) 71. Hiasa, Y., Konishi, Y. , Kamamoto, Y , Watanabe, T. , and Ito, N. Gann 62. 239 (1971). 72 Brada, Z , and Bulba, S. Res. Commun Chem Pathol Pharmacol. 3_, 383 (1972). 73. Sidransky, H , and Verney, E J Mat. Cancer Inst 44_, 1201 (1970). 74 Fare, G. Brit J Cancer 20, 569(1966). ------- 568 75 Kamamoto, Y. , Makiura, S , Sugihara, S. , Hiasa, Y. , Arai, M , and Ito, N. Cancer Res. 33, 1129(1973) '^ 76, Sidransky, H. , Ito, N. , and Verney, E J Nat Cancer Inst 37, 677 (1966). 77. Farber, E. , and Ichinose, H. Cancer Res. 18, 1209(1958) 78. Miyaji, H , Nishi, H , Watanabe, S. , Koyama, K , Tamura, K. , Nasu, K , Kusaka, H, , and Ishihama, S. Gann 48, 585 (1957) 79. Takarmya, K , Chen, S.H , and Kitagawa, H Gann 64, 363 (1973). 80. Benson, W. R. Amer J Pathol 29, 491 (1961) 81. Denda, A , Inui, S , Sunagawa, M , Takahashi, S , and Konishi, Y Gann 69, 633 (1978). 82. Konishi, Y , Denda, A., Miyata, Y , and Kawabata, H Gann 67, 91 (1976) 83 Ito, N , and Farber, E. J. Nat Cancer Inst _37, 775 (1966) 84. Miller, E.G., Miller, J.A., and Brown, R. R. Cancer Res 12, 282 (1952). 85. Miller, E C. , Miller, J.A., Brown, R.R., and MacDonald, J. C. Cancer Res 1_8, 469 (1958). 86. Hoch-Ligeti, C. , Argus, M. F. , and Arcos, J. C. J. Nat. Cancer Inst. 40, 535 (1968) 87. Marugami, M, , Ito, N , Konishi, Y., Hiasa, Y , and Farber, E Can- cer Res 2_7, 2011 (1967). A.M A. 88 Dunn, W.^C—yTTfgtf Pathol 83^, 258 (1967) 89 Svoboda, D , and Higginson, J. Cancer Res 28, 1703(1968) ------- 569 90. Chu, K. C. Chm. Med 12, 369(1965) 91. Levine, M. , and Tarver, H. J. Biol. Chem 192. 835(1951) 92. Fitzgerald, P. J. , and Hellman, L. Lab Invest 10, 2(1961). 93. Brada, Z. , Bulba, S. , and Cohen, J Cancer Res 35, 2674 (1975) 94. Farber, E. , and Magee, P.M. Biochem. J. 76, 58P(1960). 95. Stekol, J.A., Weiss, S. , and Somerville, C. Arch Biochem Biophys 100, 86 (1963) 96. Steele, R.D. Fed. Proc. 38, 382(1977) 97. Steele, R D. , and Benevenga, N J. Cancer Res 39, 3935 (1979). 98. Steele, R.D., and Benevenga, N J J. Biol Chem 254. 8885 (1979) 99. Smith, R.C. , and Pollard, D. R Biochim Biophys A eta 184, 397 (1969) 100 Smith, R.C. , and Beeman, E. A. Biochim. Biophys Acta 208, 267 (1970). 101. Jensen, D. , Chaikoff, I. L. , and Tarver, H. J Biol Chem/ 192. 395 (1951) 102. Okuzono, H. , Watanabe, S. , Serikawa, H. , and Nakana, N Kumamoto Med J 9., 61 (1956) 103. Cantoni, G. L J Biol. Chem 189, 745(1951) 104. Farber, E. , and Castillo, A E. Fed. Proc -22, 370(1963) 105. Farber, E. , Shull, K H. , Villa- Trevino, S. , Lombardi, B , and Thomas, M. Nature 203, 34(1964) 106 Rama, A , Janne, J , and Sumes, M Acta Chem Scand 18, 1804 (1964) ------- 570 107 Smith, R.C. , and Salmon, W. D. Arch Biochem Biophys. Ill, 191 (1965). 108. Stekol, J.A. , Weiss, S , Anderson, E. I. , and Toporek, M. Abstracts 132nd Meeting Amer Chem. Soc , New York, 1957, p 57C 109. Mudd, S. H , and Cantoni, G. L. J. Biol. Chem 231, 481 (1958). 110 Stekol. J.A., and Weiss, K. J. Biol Chem 185, 577(1950). 111. Wmnick, T. , and Winnick, R. E. Nature 183, 1 4ft6 (1959). 112 Stekol, J.A., Mody, U , Bedrak, E , Keller, S. , and Perry, J. Fed. Proc. j_9, 37 (I960). 113. Villa-Trevino, S. , Shall, K. H. , and Farber, E. Fed Proc 22, 237 (1963). 114. Farber, E , Lombardi, B. , and Castillo, A E. Lab Invest 12, 873 (1963). 115 Notori, Y , Trowbridge, H. O. , Toreson, W. E. , and Tarver, H. J_ Biol. Chem 236, 2821 (1961) 116. Shultz, R. L, , and Blattman, J. E Proc Soc Exp. Biol Med. 116, 604 (1964) 117. Hansson, E , and Garzo, T. Experientia 1 7, 501 (1961) 118 Hansson, E , and Garzo, T. Biochim Biophys Acta 61, 121 (1962) 119. Gross, D , and Tarver, E. J. Biol. Chem 217, 169 (1955) 120 Yoshida, A , and Yamazaki, M Biochim Biophys Acta 34, 158(1959) 121 Rabmovitz, M , Olson, M. E. , and Greenberg, D. M. J. Biol Chem 227, 217 (1957) ------- 571 122. Berg, P. J. Biol. Chem. 222, 1025 (1956) 123. Glenn, J. L, Arch. Biochem. Biophys. 95, 14(1961). 124. Zamecnik, P. C., Stephenson, M. L. , and Scott, J.F. Proc Nat Acad. Sci. US 46, 811 (I960). 125. Farber, E. , McConomy, J , Frazen, B. , Marroqum, F. , Stewart, G.A., and Magee, P. N. Cancer Res. 27, 1761 (1967) 126. Stekol, J.A., Mody, U. , and Perry, J. J. Biol Chem 235, PC59 (I960). 127. Miller, J.A., and Miller, E.G. Adv Cancer Res 1, 339(1953) 128. Orenstein, J. M., and Marsh, W. H. Biochem J 109, 697(1968). 129. Brookes, P., and Lawley, P. D Biochem J 80, 496 (1961) 130. Magee, P.N., and Farber, E. Biochem J 83, 114(1962). 131. Marroqum, R. F. , and Farber, E. Biochim.. Biophys. Acta 55, 403 (1962) 132. Ortwerth, B.J., and Novelli, G.D. Cancer Res. 29. 380(1969) 133 Swann, P. F. , Pegg, A. E. , Hawks, A , Farber, E. , and Magee, P. N. Biochem J. 123, 175 (1971). 134. Farber, E. , McConomy, J., and Fnmansky, B. Proc. Amer Assoc. Cancer Res Z, 16 (1967) 135. Miller, J.A., and Miller, E C. Lab. Invest' 15, 217(1966) 136. Natori, Y. J Biol Chem 238, 2075 (1963) 137. Borek, E., and Kerr, S. L. Adv Cancer Res. 15, 163(1972) 138. Axel, R , Wemstem, I.E., and Farber, E. Proc Nat Acad Sci US 58, 1255 (1967) ------- 572 139. Agarwal, M. K. , a ad We ins te in, I.E. Biochemistry 9, 503(1970). 140. Weinstem, I.E., Grunberger, D. , Fujimura, S. , and Fink, L M. Can- cer Res. 31, 651 (1971). 141. Kuchino, Y , and Borek, E. Nature 271, 126 (1978) 142. Hancock, R. L. Cancer Res. 31, 617(1971) 143. Pegg, A.E. Biochem J. 128, 59(1972). 144. Rosen, L Biochem. Bfophys. Res Comman._ _33, 546 (1968). 145 Sharma, O.K., Kuchino, Y. , and Borek, E. Adv. Enzyme Regulation 16, 391 (1978). 146. Yamane, Y , Sakai, K. , Shibata, M. , and Chiba, K. Gann 68, 713 (1977) 147. Craddock, V M. , and Henderson, A R. Cancer Res 38, 2135(1978) I 148. Cox, R., and Irving, C. C. Cancer Res 37, 222(1977) 149 Borek, E , and Snnivasan, P.R. Progr Nucleic Acid R es 5, 157 (1969). 150. Boehm, T. L. J. , and Drahorsky, D. Europ. J. Cancer 15, 1167(1979) 151. Jehnek, W , and Darnell, J. E. Proc. Nab. Acad. Sci. US 69, 2537 (1972). 152. Wallace, B. , and Kass, T. L. Genetics 82, 139(1976) 153 Hancock, R. L. , Forrester, P. J. , Lorscheider, F. L. , Lai, P. C, W., and Hay, M. In "Oncodevelopmental Gene Expression" (W. H Fishman, and S Selt, eds. ). Acad Press, New York, 1976, p 247 154 DeLange, R. J , and Smith, E. L Ann Rev Biochem 40, 279 (1971) ------- 573a 155. Allfrey, V G. , Faulkner, R. , and Mirsky, A. E. Proc Nat Acad Sci. US 51, 786 (1964). 156. Paik, W. K. , and Kirn, S. Biochem. Biophys. Res Comirmn 29» 14 (1967). 157. Friedman, M. , Shall, K H., and Farber, E. Biochem Biophys. Res Comrnun 34, 857 (1969). 158. Baxter, C.S., and Byvoet, P. Cancer Res 34. 1418(1974) ------- 573b SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.2.1.5 1. Farber, E.: Adv. Cancer Res. 7, 383-474 (1963). 2. Stekol, J.A.: Formation and Metabolism of S-Adenosyl Derivatives of S-Alkylhomocysteine in the Rat and Mouse. In "Transmethylation and Methionine Biosynthesis" (S.K. Shapiro, and F. Schlenk, eds.) University of Chicago Press, 1965, Chapter 14, p. 231-248. 3. Fitzgerald, P.J. and Hellman, L.: Lab. Invest. 10, 2-30 (1961). 4. Sharma, O.K., Kuchino, Y., and Borek, E.: Adv. Enzyme Regulation 16, 391-405 (1978). ------- NOTES ADDED AFTER COMPLETION OF SECTION 5.2.1.5 Purchase et al. (1) have recently demonstrated the in vitro carcino- genicity of ethionine using a mammalian cell transformation assay. Positive results have also been obtained in two other predictive tests - degranulation of rat liver endoplasmic reticulum, and tetrazolium reduction. An unpublished observation by Weisburger ^t_ jl_. (cited in ref. 2) reveals that although ethionine itself is not mutagenic in the Ames test, its vinyl analog is highy mutagenic. Considering the known activation pathways of ethionine (see Section 5.2.1.5.4), the authors (2) speculate that the mutagenic and carcino- genic actions of ethionine may involve the production of an epoxide (epoxy- homovinylcysteine) via vinylhomocysteine. Cox and Tuck (3) have recently shown that ethionine inhibits the methylation of lysine and arginire residues of rat liver histone in vivo possibly via accumulation of S-adenosyl-L- ethionine. The importance of this effect in ethionine carcinogenesis remains to be elucidated. It has been suggested (4) that these methyl groups are involved in establishing the higher order of chromatin structure by inter- acting with neighboring proteins. References for Section 5.2.1.5 Update 1. Purchase, I. F. H., Longstaff, E., Ashby, J., Styles, J. A., Anderson, D., Lefevre, P. A., and Westwood, F. R.: Bf. _J. Cancer 37, 873 (1978). 2. Weisburger, J. H., and Williams, G.: Chemical Carcinogenesis. _In_ "Casarett and Doull's Toxicology" (J. Doull, C. Klaassen, and 11. Amdur, eds.) Chapter 6, MacMillan, New York, 1980, p. 84. 1 ------- 3. Cox, R., and Tuck, M. T.: Cancer Res. 41, 1253 (1981). 4. Duerre, A., and Quick, D. P.: Rat Brain Histone Lysine Methyltrans- ferase. _In_ "Transmethylase" (E. Usdin, R. T. Borchardt, and C. R. Graveling, eds.) Elsevier/North Holland, New York, 1979, p, 583. ------- |