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

-------
                                                                        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








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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)

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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


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146.  Yamane,  Y ,  Sakai,  K. ,  Shibata, M. ,  and Chiba, K.   Gann 68,  713


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147.  Craddock, V  M. , and Henderson, A  R.  Cancer Res  38,  2135(1978)
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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


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      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)

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                                                                    573a







155.  Allfrey,  V G. , Faulkner, R. , and Mirsky, A. E.  Proc  Nat  Acad




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                                                                      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).

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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

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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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