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VOLUME 31 NUMBER I *"TN*>UW>Oy
I JANUARY ( 1982 ,
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Biochemical Phaimacology
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CONTENTS
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Commentary
II. M. Bolt, R. J. Lahi and J. G. Filsek
I Reactive mclabolilcs and carcinogenicity of halogcnalcd cihylcncs
Research Papers
Matthew M. AMi.snnd Sandra K. Fkank
5 Slcrcoclicinical aspects of /iitra-chloroamphetaminc metabolism. Rabbit liver microsomal metabolism of
RS-, /<( -- )-, and 5(+)-pora-chloroamphclaminc
F, Kibs/.G, L. I. Horvath, N. Simon, Cs. Sikl6si and M. Kiss
11 The role of possible membrane damage in porphyria cutanea tarda: a spin label study of rat liver cell mem branes
Maktyn T. Smith. HjOrdis Thor. Pia Hartze_l and Sten Orrfnius
19 The measurement of lipid peroxidation in isolated hepalocytes
David E. Clarke, Geoffrey A. Lylfs and Brian A. Calunoiiam
27 A comparison or cardiac and vascular clorgylinc-
rcsistant amine oxidase and monoamine oxidase. Inhi bition by amphetamine, mcxilcline and other drugs
Russell Hilf and Deirdre L. Helton
37 Reduction of proline transport in R3230AC mammary carcinomas by estrogens in vitro
David M. Helfman and J. F, Kuo
43 Differential effects of various phosphodiesterase inhi bitors, pyrimidine and purine compounds, and inorganic phosphates on cyclic CMP, cyclic AMP and cyclic GMP phosphodiesterases
Inca Mounsf.y, Karen A. Brady, Jacky Carroll, Robert Fisher, and Derek N, Middlemiss
49 K*-cvokcd [1H)-5-HT release from rat frontal cortex slices: the effect of 5-HT agonists and antagonists
S, Akiinoukii, N, El-Shazly, N. Sallam, S. El-Meleoy, S. M. El-Sewedy, M, H. Mostafa and E. A. El-Uassiouni
55 In nit'll clfcct of chloramphenicol and thiamphcnicol on some enzymes of normal mouse liver
Continued on outside bock cover
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COMMENTARY
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REACTIVE METABOLITES AND CARCINOGENICITY OE HALOGENATED ETHYLENES
H. M. Him r. R. J. Laid and J. G. Fu ser I'harniakologisches hisiilui. Ahtcilung Toxikologic. Johannes Guienhcrg-t'nr.eriita! Mam/. (there
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Oxirancs ni biological reactive intermediates of huloetltylcnes
A field of rapidly expanding knowledge is that of
biological reactive intermediates. In ibis discussion halogenutcd compounds, e.g. vinyl chloride, play a prominent role |1|. For the chlorinated cthylen'es. preliminary considerations connecting structure and activity have been published by Henschler and co workers [2-5]. They suggested the following: (a) all chlorinated ethylenes are initially biotransformed by microsomal monooxygenase (si to epoxide l oxirane) intermediates which are subsequently `detoxified', e.g. by molcculai rearrangement (see Fig. 1): (b) reactivities and hence loxicitics of individual chlorooxirane intermediates depend on the type of chlorine substitution in that symmetric substitution renders the epoxide relatively stable ad not mutagenic whilst asymmetric substitution causes unstable and, therefore, mutagenic epoxides: (c) trichloroethylene is exempt from this general rule because us epox ide 2,2.3-trichlorooxiranc, although asymmetrically substituted and reactive, is immediately further transformed at the cytochrome IM.sd site to trichloroacetaldehydc (chloral).
The latte, is now supported by a recent inhalatory carcinogenicity study in 3 animal species which revealed no indication for carcinogenicity of tri chloroethylene [6|. Also, mutagenicity data orig inally obtained with F.. coli K 12 [3| were generally
confirmed by using S. typhimurium strains (7|. This study by Bartscb el at. (7) also included one hrominnted and one lUiormated ethylene, vinyl hiotmde and vinylidene fluoride; both asymmetrically substi tuted halocthylenes were hioactivatcd to mutagenic metabolites, hut to much different extents. Vinyhdene fluoride showed only `marginal mutagenicits '.
Covalent binding, mutagenicity and carcinogenicity
During the last few years more data became avail able on covalent binding of haloelhylene metabolites to proteins and to nucleic acids; carcinogenicity bioasstiys were also performed. Furthermore, the pharmacokinetics of these compounds has been sys tematically investigated. This now extends the data base for a reasonable comparison.'
A compilation of some relevant literature data (8-311 is given in 'fable 1. Along with the current opinion that epoxides are essential intermediates in metabolism of these compounds, all the halocthylencs in which this effect has been studied are in fact biotransformed to protein alkvlating intermediates (2. 8, lJ, 15, 16. 21. 22, 25. 26.'2y|. However, alky lation of nucleic acids is not a common feature as pcrchloroethylene (2b|. ami probably trichloroeth ylene (as discussed in |24|). do not lead to defined alkylation products at nucleic acid bases, much in contrast to compounds like vinyl bromide |3()|, vinyl chloride (1(>--12) ami vinylidene chloride (17. 32|.
faille 1. Cmalcnt iiiaenunolcailar hunting ol meialvulues, nuiiagemcni and carcmoecmeuv ol h.doi>enaicd ethvleues
1 lulocihylcnc
Covalent protein binding
in ntru in oir*i
Covalent hmdmi! Mutagenicity in bacterial
to nucleic acids
tests alter
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anmial hioussays
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V tin Yinvl chloride
, ii'| 1 Vinvlidcne chloride K, /; ys' 1,2-ii.v-DichUiroeilivleiie Y <1 C /1.2-iniJi.i-Diehloroelhvleiie VC'JL^J'JW Trichloroethylene iv >6-71 I'eiehloioethylene Vt-TiJ4! ^ tn>l bnuniu'e
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t i-iposarcomas reported in rats alter oral adnunisiratjon. the biological siisnilieaiKv oi which k Mill not clear No specific alkylation products identified |23],
II Not carcinogenic in rats (27, 2N|; hepaiomas m IKOR mice alter high oral lineage |2x|. die luologieal sigmlieanee
of which bcinj! subject of present discussion (25,26} T| Low degree of alkylation re|Mined.
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This is consistent with I Icnschlcr's structural theory, and it lends biuehentical support lo the outcome of both mutagenicity |3. 7| and carcinogenicity [6. 13. 14, IH-20,27.28.3l]lests, Hence, there arc different lines of support (metabolism and covalent binding, mutagenicity, carcinogenicity) to the view [33] that epoxides of halogenatcd cthylcnes in fact must pos sess a certain degree of instability to effect a genotoxie action.
On the other hand, it seems feasible that an epox ide, being too unstable to reach the DNA target in significant amounts, could even be less active than a more stable one. Quantitative data interpretahle m this direction have now been accumulated.
Metabolic rales anti formation ofpreneoplastic hepa tocellular foci
Recent publications have impressively demon strated that metabolic and pharmacokinetic data must be incorporated into the interpretation of tox icological data, especially when inhalation experi ments are concerned |34,35). Therefore, in view of the very dissimilar metabolic rates of lialoethylcnes, it is mandatory to base a mechanistic and quantitative comparison of oncogenic effects on metabolic data, i.e. on the rates or amounts of intermediary epoxide produced by the initial metabolic step (Fig. I). There is sufficient pharmacokinetic data (36, 37] for such calculations.
Oncogenic effects of different haloethylenes can
be quantitated on the base of histochemical exami nation of pre neoplastic nucleoside-S'-triphosphatase ("ATPasc") deficient foci in rats exposed lo these chemicals from the time of birth on: a detailed dis cussion of this mcthodologv has been published |3,S.
Mi-
Table 2 contains metabolic data [36. 37J and data on formation of hepatic preneoplastic foci [23. 3R-43] after exposure of young NVistar rats lo halocthvlenes. A suitable endpoint for such a comparison [39] is the percentage of ATPasc deficient hepatocytes after an exposure period of It) weeks. The exposure concentrations of 2001) ppin ensured a satu ration of metabolizing enzymes (t'V,, conditions) [36|; only vinylidene chloride, because of its high acute toxicity, was applied at a concentration that produced a metabolic rate of about } IV., [37). In accordance with Jfenschler's general rules (<Wc
supra) trichloroethylene |3K| as well as perchloroethylcne [25. 39] do not induce preneoplastic foci. The other compounds show a wide range of onco genic activities, extended over 3 orders of magnitude with the extremes of vinyl chloride |3b] and vinyhdene fluoride |41). This huge range is much reduced when the observed preneoplastic effect is related to
the amount of haloelhylene metabolites produced under the experimental exposure conditions (400 hr exposure; last column of Table 2). Such an estimation is realistic because newborn rats develop their full ability to metabolize vinvl chloride within their first week of life [44].
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Fig. 1. Initial steps of metabolism of halogcnatcil eihylenes [2-5. 7. II).
Table 2. Mciahofi.'*
I lalocthylcnc
Vinyt tluoride Vinylidene lluorii Vinyl chloride Vinylidene chluwl' j Trichloroethylene IVrChloroethylcni: Vinyl tsromide
Under the ns essential initial' halooxirancs, tk determined by { cquivaleni to the > the epoxide is i f carcinogenic) ptj for vinyl chloric data in the last cl1 idea of the cor; different epoxid: Front this compi-s
(A) The oncx] metabolites d" chloride > vinyl are available o bromide subs physico-cbemi although the i olism of vinyl bi [7]. But comp been publishe ]46): these shy] fluorooxirane h than that of chi
(B) When thefj haloethylenes (i idc and vinylid compared (Tabj intermediates tinctly lower O monohaloethyli are available orf idene fluoride, rune (the epoxitj ably more unsta the former, byj attempts of syn
These two pi tradict Hcnschl that instability oxiranes in gen and carcinogen ylenes. Witho reactive to suco structurally cloj w-ide range off halogenatcd et)
Reactive metabolites and carcinogenicity of halogenated eihslcnes
Table 2. Mclabolic rales of halocihslcncs and ATPase deficient prcncoplastic foci observed afier lb weeks of exposure (Xhr/dav; 5 dasVwcek) in newborn female Wisiar rais
Halocthylene
Vinyl fluoride Vinylidenc fluoride Vinyl chloride Vinylidene chloride Trichloroethylene Pcrehloroclhylene Vinyl bromide
Exposure concentration
(ppm)
Metabolic
rate {tm\a\c\ Ur-kg/
Intimated amount metabolized during
exposure (mmolc/k)!)
200d 2000 2000
100 201X1 2000 20<XI
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14.3 1 Kl IK 2 It,15 0 0 4 }K
Under the assumption [2-5J of Fig, 1 that the
essential initial metabolic step is transformation to
halooxirancs, the metabolic rates of haloethylenes determined by pharmacokinetic means must be equivalent to the amounts of epoxide produced. That the epoxide is in fact the ultimately reactive (and carcinogenic) principle has recently been validated for vinyl chloride |45], Hence, comparison of the data in (he last column of `f able 2 should give some idea of the comparative oncogenic effects of the different epoxides under realistic conditions in vino. From this comparison, two features may be deduced:
(A) The oncogenic effects of monohnloethylenc metabolites decrease in the order: vinyl chloride > vinyl fluoride > vinyl bromide. No details
are available on the metabolic pathways of vinyl bromide subsequent to epoxidation and on physico-chemical characteristics of its epoxide, although the implication of this epoxide in'metab olism of vinyl bromide has been reasonably suggested [7], But comparative molecular orbital studies have been published on lluorooxiranc and chlorooxirane [46]; these show that the 3-membercd ring of fluorooxiranc has more tension and is less stable
than that of chlorooxirane. (B) When the 1,1-dihaloethylenes and the mono-
haloethylcnes (i.e., vinylidene fluoride vs vinyl fluor ide and vinylidenc chloride vs vinyl chloride) are compared (Table 2), it appears that the reactive intermediates of 1,1-dihaloethylenes exert a dis tinctly lower oncogenic effect than those of the monohalocthylencs. Whilst no experimental reports are available on a hypothetical [7] epoxide of vinyl idene fluoride, it is known [5] that 2,2-dichlorooxirane (the epoxide of vinylidene chloride) is consider ably more unstable than monochlorooxiranc; hence, the former, by contrast to the latter, "resists all
attempts of synthesis by conventional methods" [5],
These two points, tit a first glance, seem to con tradict Henschler's structural rule [2-5] which says that instability of chlorooxirans (and possibly halo oxirancs in general) is associated with mutagenicity and carcinogenicity of the parent halogenated ethylenes. Without any doubt, epoxides need to be reactive to successfully attack the DNA target. Also, structurally closely related epoxides can exhibit a wide range of genotoxic activities [47], For the halogenated ethylenes, however, it appears that the
epoxide of vinyl chloride, monochlorooxiranc. in quantitative terms might represent an optimum between stability and reactivity to both reach the DNA target and react with it. after being formed at the monooxygenase site. A further decrease in sta bility could possibly render the oxirane too short lived to reach the target.
Future experimental and theoretical work should be directed to these questions. The presently avail able data demonstrate that the haloethylenes rep resent a family of compounds of very dissimilar bio logical activities although their simplicity in chemical structure implies a certain uniformity as to the pos sible routes of metabolic transformation. Hence, ideas to broaden the molecular and theoretical basis of understanding the differences in biological effects
must be encouraged.
Acknowledgement--The authors' work has been financially supported by the "Deutsche Forsehungsgemeii.schafi" (Grant No, Bo 491/3).
KKFKKKNCKS
1. R. Snyder, D. V. Parke. J. Kocsis. D. J. Jnllow and Ci. G, Gibson I Eds), Biological Reactive Intermediates 2: Chemical ,1/cciuintnm ami Biological Ejfectx. Plenum Publishing Corporation. New1 York (19X1)
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e w Oro ro
oo
ro
if
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