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
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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
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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)
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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
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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
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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) .
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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
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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,
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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)
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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.
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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
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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
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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
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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
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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
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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
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N-ocetylethionme sulfoxide
Ethionme sulfoxide
Oxidation
(11)
Sulfur
activation
ATP-
Corboxyl activation
S-odenosylethionme
Transethylation
Ethyl derivatives of
macromolecules
Figure 28
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LEGEND TO FIGURE 28
Fig. 28. Basic metabolic pathways of ethionine
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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
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LEGEND TO FIGURE 29
Fig. 29. Transaminative pathway of ethionine catabolism.
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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
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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
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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
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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)
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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
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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).
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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.
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