Document O3LxmQym4XEnDYX24k1Yo7M8v
Tetrachloroethylene Trichloroethylene 1.1-Dichloroethylene 1 2-Dichloroethylene 1 Vinyl Chloride
1977 file: K-2521 -d 00)
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K-2520-
1977
K-2197-
1977
K-2517K-1711-
1977
Vol. 21. pp. 61-64. 1977
Metabolism and Mutagenicity of Halogenated Olefins--A Comparison of Structure and Activity
by D. Henschler*
Chlorinated t'thxlrncs are metabolized in mummuk, ns a first step, to epoxides. The fate of these
electrophilic intermediates niav be reaction with nucleophiles (alkylation), hydrolysis, or intramolecular rearrangement. The latter reaction has been studied in the whole senes of chlorinated epovicihanes. The
rearrangement products found were: acyl chlorides (tetrachloro-, trichloro-, and 1,1-dichloroethylenes), or chlorinated aldehydes (1.2-dichloroethyJenes, c,,t- and frnru*, vinyl chloride). The metabolites found m
cu'o are identical with, or further derivatives of these rearrangement products, with one important
exception: trichloroethylene. With this compound, in vivo rearrangement yields chloral exclusively. The
mechanism of the different rearrangement has been identified as a Lewis acid catalysis. All chlorinated elhylene.s have been investigated in a tissue-mediated mutagenicity testing system. The
prumment molecular feature of those with mutagenic effects (trichloro-, J.l-dichloro-, and mono-
chlorocthylene) is unsymmetric chlorine substitution which renders the epoxides unstable, whereas sym metric substitution confers relative stability and nonmutagenic property.
Since vinyl chloride has been demonstrated car cinogenic in humans (!) and experimental animals LZ). and further reports suggested carcinogenic pioperties of the chemical congeners vinvlidene chloride (_?, 4) and trichloroethylene p), we became interested in the question whether or not other members of the group of chlorinated cthylcnes would share this type of biological activity, and what would be the molecular features or prerequi sites. respectively, which are responsible for the carcinogenic effect. We started with a theoretical consideration of the chemical activity of the molecules which focused on the influence of the chlorine substitutions. In a second step, the metabolic bioactivation and degradation was studied in vivo and compared with the theoretical expectations from the chemical reactivity. After this, the mutagenic activity was investigated in vitro in a modified Ames testing system. Finally, the re sults of this mutagenicity test were compared with the in' vivo carcinogenicity findings as reported by others, and > tentative mle of chemical reactivity, biot ran summation and mutagenicit y/caieinogenicuy was worked out.
Inxtitui Uir Toxikiilugic taut Pharrna^oloLiic, UniverMaii Wurzburg. D-S7no Wurchun:. West Gerrruny.
Chemical Reactivity
The chemical reactivity of chlorinated alipha'ie compounds is decisively determined by the chlorine subsiitution(s). However, the influence is com pletely different in alkanes, alkenes, and alkynes. In general, chlorine substitution exeits a stabilizing effect on account of steric protection by the bulky substituent. This interferes with the electronwithdrawal effect of the chlorine substituent. In the case of chlorinated alkanes, the result is a destabili zation of C-C and C-CI bonds. C-C and C-CI fis sions under formation of free radicals are conse quent reactions in the metabolic conversions of C, or C- compounds.
A quite different influence is encountered with chlorinated ethylenes. In this class of molecules, the electron-withdrawal effec dominates over the mesomeric donator ellect ot the involved carbon atom, thus decreasing the electron density in the double bond which in turn results in a chemical stabilization against eleetiophilie attacks (6). The stabilizing effect increases with the number of chlorine substitutions. It is optimal m tetrachlorocilnleno. as has convincingly been demonstrated by the reactivity of chlorinated etfnlenes with ozone (7).
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In alkynes, chlorination results in a destabiliza
tion. as has been shown with mono- and dichloroacctylene. which are considerably less stable than acetylene (8).
Metabolic Activation and
Deactivation of Chlorinated
Ethylenes
The first step of metabolic transformation of chlorinated ethylenes in mammals is epoxidation. Up to now there is onl\ indirect evidence for this reaction, (a) by the demonstration of a specific bind ing spectrum with P-450 in the case of trichloro ethylene (9), and (b) by the type of metabolites iden tified in vitro as well as in vivo.
In general, chlorinated epoxides may undergo a variety of reactions (see Fig. I): reaction with nu cleophilic cellular macromolecules under alkyla tion: conjugation with low molecular nucleophiles (mainly glutathione) both enzymatically and nonenzymatically; hydrolysis to diolx. again with and without the catalytic action of enzymes (epoxide hydrase): and intramolecular rearrangement. The latter reaction represents a deactivation mechanism and is of considerable importance for the potential of acute toxicity as well as of carcinogenicity and mutagenicity of the different members of the scries of chlorinated ethylenes.
r*Ott<on with cfllylor
q*iMffiuiut^Qf ntiounclroe^itu
fniyntqin "on- rrtiyfTVQt (QltatOl'X'Git* )
Cjrrorq#mfnt
-cc1Ii-c11 OH OH hy<3r Al y M \
r>on .
fniymahc )
IH Qld*hy4rt
1 Cl OCyl cbtornjp*
Figurc I. Kcuclions of ehlorinuled aliphatic epoxides niter
formation in the celt.
We have studied the chemical behavior of these epoxides which have been synthesized. The excep tion was 1,1-dichloroethylene oxide, which cannot be obtained by conventional methods, apparently because it is extremely unstable on account of its high polarity (10). They rearrange either to acyl chloride-, (tctrachloro-, trichloro-, and 1.1dichloroethylene) or to chlorinated aldehydes
cis- and /ranj-dichlorocthylene, and vinyl chloride
[Eqs. (I)-(5)J. The expected metabolites would be trichloroacetic acid (with tetrachloroethylenc), dichloroacetic acid (tri- and 1,2-cis- and transdichloroethylene) or monochloroacctic acid (vinylidenc and vinyl chloride), and derivatives of monochloroacetic acid (or chloroacetaldehydc)
after conjugation with glutathione.
The predicted metabolites have been identified in systematic experiments with the isolated perfused rat liver preparation (10), with one important ex ception: trichloroethylene. With this compound, es sentially no dichloroacetic acid was detected; this has recently been confirmed by Leibman in a mi crosomal metabolizing system (//). The different behavior of trichloroethylene oxide in vitro (thermal rearrangement to dichloroacctyl chloride) and in vivo (formation of chloral) is outlined in Figure 2.
ci ci ci- =ccVi ---
TETRACHCOROETMYIENE
lsc ,--0,c,CI
Cl N __ H
c/ Vi
trichloroethylene
on
V-Y
HH
>=< --
Cl ci Cit-l 2-OlCHLORQElHYLENC
Nc >--0N c'
H \ fci / --C\
>
Cl H
irons -1 J -OlCHLOROETHYLENE
\ ,o% / C<-?C
Cl H
>-<
--
Cl H tl-OlCHLOROE THriE NE
\c /--On c "
Cl H
C =i C H^ H
VINYL CHLORJOE
c:c ,--o^c,h
(I)
Ci
(2)
(3)
Cl (4)
CH,CI-C <5)
This difference might be of crucial significance
for the evaluation of the carcinogenic potential of trichloroethylene. We therefore studied the rear rangement mechanisms in a more detailed investi gation. Theoretically, there are three paths of in tramolecular rearrangement. A simple hydride mig
ration which would lead to dichloroacctyl chloride has low probability of occurring (12). The formation of o-kctocarbonitim ions after C-O heterolysis xvhich is the most probable path, could occur in txvo versions, resulting either in a single [Eq.
62 Environmental Health Perspectives
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f7 I
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*i chlon *oulu
ihyle: uxi iri Xld 'tjUlvc
'jiUch
.xntil' t rer
VfUf! rxxjn t;:cg ,,i in * Ja:
u/i x* j ncu*
.41
i5)
^I'oncc '"'"nii;i| of
` :'e rearimcsti-
of in-
mil;.
; '_'rm:iiion ,er<'lv\is `ur in (w0
'''lc [Eq.
c=c --
Cl^
lr*tM<xo*tnyi**
'i
n
/0\.
ci
v,,"-c-c^0 ^ 'a c'
dChlort>QctylcMOfid*
compounds. After extended preliminary studies, E. coli K 12 was found an acceptable tester strain.
The results are given in Figure 3. There was no direct mutagenic activity of the compounds. Only after metabolic activation by added phenobarbitalinduccd mouse microsomes some of the compounds
yielded positive results. The data cannot be com
Cl pared on a quantitative basis because the concen
chloral
/
CCIj-CHjOH
Inchiorooihanol
\ ^0
CClj- C'
OH
tnchtoroccrtic ac*d
trations of the test compounds varied from 0.6/mV/ (tetrachloroethylene) to I0.6mA4 (vinyl chloride). However, vinyl chloride obviously is the most active compound. Vinylidene chloride and tri-
chloroethylene exert a small but definite mutagenic
CClj-- CHjO- Glue
effect, whereas tetrachloroethylene and i,2-cis- and
I Figure 2. Formation of trichloroethylene epoxide and rear rrn/is-dichloroethylenes are inactive.
I
rangement in viiro (to dichloroacetyl chloride) and i/i Wv<j (to chloral) and further metabolic products.
Cl Cl Cl Cl Cl H
s/
-
X/
CmC^ cTMc
C*CS
MM
H Cl
C-C
H Cl
c--c
(6)] or a double chlorine-substituted carbe-
nium [Eq. (7)j; the latter (disubstituted), which
would yield chloral, is less stable and thus has the
lower probability. I The rearrangement to chloral [Eq. (7)], which is
the only path detected in vivo can be forced in vitro
by the catalytic action of strong Lewis acids like
FeCl.i or AIC1;, (10). We have speculated therefore,
that the only formation of chloral (and further oxi
dation or reduction products such as trichloroacetic
acid and trichloroethanol, and its gjucuronide) in vivo
could be due to catalytic action of the iron of P-450 in
the trivaient form at the site of the formation of the
epoxide (6): experiments with purified P^150 to con
firm or reject this assumption are in progress.
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ao h
101 H
\ .A /
c-c -- - - Cl-- c-- c
/\
,-\-J 1
Cl Cl
,CICl
---
CH CI1; ---
s Cl
(6)
Figure 3. Mutagenicity of chlorinated ethylenes in E. coli K 12 after metabolic activation by incubation with stimulated mouse liver micro$omc5 U3).
Chemical Reactivity of
Metabolically Formed Epoxides;
Cl A H \cA--- c/ ----
Cl/ \Cl
Cl 101 _C1 -- C1 -H --cil*_:-cKi
C Clj-- c
H (7)
Mutagenicity and Carcinogenicity
A view at the synopsis of the structural peculiarities of the epoxides (Fig. 4) reveals that the common feature of those which are mutagenic is an
,
Mutagenicity of Chlorinated
unsymmetric chlorine substitution, wheieas the in active compounds are characterized by a symmetric
1
Ethylenes
distribution of the chlorine atoms. There is evi
dence Irom the chemical literature that unsymmet
)
i
The six chlorinated ethylenes have been tested for ric chlorine substitution renders the epoxides un their mutagenic potential in a modified Ames stable (10, 16). as compared to symmetric substi tissue-mediated microbial system (13). SulntonvUu tution. where the epoxides are relatively stable (17.
typhimurium as tester strains, which had previously /if). The reason for the instability of an unsym
been successfully used with vinyl and vinylidene metric substituted oxirane is the preponderance of
chloride (/4, 15). were found unsuitable because of the electron withdrawal effect of the chlorine
the high primary toxicity exerted by some of the atom(s).
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December 1977
63
-$-
-A m
a ,o, ct c-cV
aa
tetroehtorocthylene
y H Li "v /'\A' aa
cts-12-dichlorotthylene
sA ah
trichloro ethylene
clvA
/c-c \
11-dichloroethylene
Ct H
^ A.a ah
tran$-l 2-dichloro ethylene
Cl 0 H
^c- cN vinylchloride
HH
symmetric rel stable not mutagenic
asymmetric unstable mutagenic
Figure 4. Tentative rule of interrelationship betvcen chemical structure, electrophilicity (stability), anu mutagenic (car cinogenic) potential of epoxides ofchlorinated ethylenes (/9).
We have deduced from these interrelationships the following tentative rule: high electrophilicity is a prerequisite for the mutagenic and carcinogenic ac tivity of the unsymmctric chlorinated ethyienes trichloro-, 1,1-dichloro-, and monochloroethylene {13, 19). So far, the predicted carcinogenicity has been confirmed by vinyl chloride (/, 2), vinylidcnc chloride (2. 4), and trichloroethylene (5).
However, the carcinogenic potential of trichloro ethylene has not yet been demonstrated convinc ingly. The NCI study (5) has been performed with a technical sample which contained strong elec trophilic epoxides as stabilizers (epichlorohydrin, epoxibutane) {20) which most probably confer the carcinogenic effect. As pointed out above, the chemical behavior of trichloroethylene oxide differs in vitro and in vivo. The probability prevails that this epoxide is completely detoxified by rearrange ment to chloral under in vivo conditions in mam mals. A solution of this problem is open to further carcinogenicity tests in whole animals and biochemical studies on the bioactivation and deac tivation of trichloroethylene under realistic expo sure conditions.
REFERENCES
1. I.A.R.C. Ini. Tech. Rcpi. on X inyl Chloride, 75/001. Lyon. 1975.
2. Maltoni. C.. and Lefeminc. G. Carcinogenicity bioassays of vinyl chloride. I. Environ. Rev 7: 387 (1974).
Viola, P. L.. cited by Bartsch, H., et at. Nature 255: 641 (1975). Maltoni. C. Paper presented at International PVDC Semi nar, Hamburg, Jan. 26. 1977.
DHEW. Memorandum on Trichloroethylene. Dept, of Health, Education and Welfare, Washington. D. C., 1975.
Bonse, G., and Henschler, D. Chemical reactivity, biotransformation, and toxicity of polychlorinated aliphatic compounds. CRC Crit. Rev. Toxicol. 4: 395 (1976).
Williamson, D. G.. and Cvetanovic, R. J. Rales of reactions of ozone with chlorinated and conjugated olefins. J. Amer. Chcm. Soc. 90: 4248 (1968).
Ott. E. Uber das Dichloroacetylen. Chem. Bcr. 75: 1517
(1942).
Uchlcke, H, el at. Spectral evidence for 2,2.3-trichlorooxirane formation during microsomal trichloro ethylene oxidation. Naunyn-Schmiedebergs Arch. Pharma col. (Suppl.) 253 (1976).,
10. Bonse, G., ct al. Chemical reactivity, metabolic oxirane
formation and biological reactivity of chlorinated ethylenes in the isolated rat liver preparation. Biochem. Pharmacol. 24: 1829 (1975).
II. Lcibman. K. C.. and Ortiz. E. Microsomal metabolism of chlorine ethylenes. Paper presented at 6th International Congress on Pharmacology. Helsinki, 1975: Abstr. 257: No. 608.
12. McDonald, R. N.. and Schwab, P. A, Molecular rear rangements. II. Chlorine migrations in the epoxidc-carbonyl rearrangement. J. Amer. Chem. Soc. 85: 40(M (1963).
13. Greim. H.. et al. Mutagenicity in vitro and potential car cinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol. 24: 2013 (1975).
Rannug, V., et al. The mutagenicity of vinyl chloride after metabolic activation. Ambio 3: 194 (1974),
Bartscb. H.. Malavcille, C., and Montesano. R. Human, rat, and mouse liver-mediated mutagenicity of vinylchloridc in S. Typhimuni'm strains. Int. J. Cancer 15: 429 (1975).
Gross. H.. and Freiberg, J. Zur Existenz von Chlorathylenoxid. J. Prakl. Chem. 311: 506 (1969).
Frankel. D. M.. Johnson, C. E,, and Pitt, H. M. Prep aration and properties of tclrachloroethylene oxide. J. Org. Chem. 22: 1119 (1957).
IS. Griesbaum. K., Kibar, R., and PfelTer. B. Synthese und Stabilitat von 2.3-Dichloroxiranen. Justus Liebigs Ann. Chem. 1975: 214.
19. Henschler. D.. Bonse, G.. and Greim, H. Carcinogenic potential of chlorinated ethylenes--tentative molecular rules. Paper presented at 3rd WHO-IARC Meeting, Lyon.
Nov. 3. 1975.
Henschler. D.. ct ai. Carcinogenicity of trichloroethylene: lact or artifact? Arch. Toxicol.. 37: 233 (1977).
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