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V {orne-i^an R&S 115640 ft.'wrmteol Ptormocoiw- V1 31. No, 1. pp 1-4. 1982 primed m Ori* Bntam. iWe^?5r K (UMU 3 Per^mon Press Ltd COMMENTARY REACTIVE METABOLITES AND CARCINOGENICITY OF HALOGENATED ETHYLENES H. M. Bolt, R. J. Laib and J. G. Filser Pharmakologisches Institut, Abteilung Toxikologie. Johannes Gutenberg-Universit&t Mainz. Obere Zahibacher StraQe 67, D-6500 Mainz. West Germany r, <4 f - ' ! . /' />/ i- BBJmsgfe; biological reactive intermediates of confirmed by using S. typhimurium strains [7], This haloethylenes study by Bartsch et al. [7] also included one brom- A field of rapidly expanding knowledge is that of biological reactive intermediates. In this discussion halogenated compounds, e.g. vinyl chloride, play a prominent role [1]. For the chlorinated ethylenes, preliminary considerations connecting structure and inated and one fiuorinated ethylene, vinyl bromide and vinylidene fluoride: both asymmetrically substi tuted haloethylenes were bioactivated to mutagenic metabolites, but to much different extents. Vinyli dene fluoride showed only `marginal mutagenicity'. activity have been published by Henschler and coworkers [2-5]. They suggested the following: (a) all Covalent bindineBgtgffffBB&fid carcinogenicity 1 chlorinated ethylenes are initially biotransformed by During the last few years more data became avail microsomal monooxvgenase (s) to epoxide (oxirane) able on covalent binding of haloethylene metaboli^^ intermediates which are subsequently 'detoxified', to proteins and to nucleic acids: carcinogenic^y e.g. by molecular rearrangement (see Fig. 1): (b) bioassays were also performed. Furthermore, the] reactivities and hence toxicities of individual chloro- pharmacokinetics of these compounds has been sys-' oxirane intermediates depend on the type of chlorine tematically investigated. This now extends the data substitution in that symmetric substitution renders base for a reasonable comparison. epoxide relatively stable and not mutagenic A compilation of some relevant literature data ilst asymmetric substitution causes unstable and, [8-31] is given in Table 1. Along with the current therefore, mutagenic ffiimnBB (c) trichloroethyleneopinion that epoxides are essential intermediates in is exempt from this general rule because its epoxmetabolism of these compounds, all the haloethy ide 2,2,3-trichlorooxirane, although asymmetricallylenes in which this effect has been studied are in fact substituted and reactive, is immediately further biotransformed to protein alkvlating intermediates transformed at the cytochrome P-450 site to tri- [2, 8, 9, 15, 16, 21, 22, 25, 26.'29]. However, alky chloroacetaldehyde (chloral). lation of nucleic acids is not a common feature as The latter is now supported by a recent inhalatory perchloroethylene [26], and probably trichloroeth carcinogenicity study in 3 animal species which ylene (as discussed in [24]), do not lead to defined revealed no indication for carcinogenicity of tri alkylation products at nucleic acid bases, much in chloroethylene [6], Also, mutagenicity data orig contrast to compounds like vinyl bromide [30], vinyl inally obtained with . coli K 12 [3] were generally chloride [10-12] and vinylidene chloride [17. 32]. Table 1. Covalent macromolecuiar binding of metabolites, mutagenicity and carcinogenicity of halogenated ethylenes Haloethylene Covalent protein binding in vitro in vivo Covalent binding Mutagenicity in bacterial to nucleic acids tests after Carcinogenicity in in vitro'; in vivo+ metabolic activation animal bioassavs JT?^t^Orattt)etftylene PerchlorocUrviene- -- X - |81 X X X -[211 -[251 -[29] X -[9] + [15.16] X X -[22] + [2.26] X X * [10-12]*.* -<(17]+ X X -.-*[23.24; - [26|+ - [30!*.+ X (-H7] - 13- 7] + [3.7] -[3] -PI ( + ) [7] -13.7] * [7] X *t|13] -[14] +/- [18-20] X X -|6] -ii [27.28] -[31] - = positive. - * negative: x = not vet reported: (-) = "marginal mutagenicity" [7]: J-- positive and negative reported. m Liposarcomas reported in rats after oral administration, No specific alkylation products identified [23]. the biological significance of which is still not clear. Not carcinogenic in rats [27.28): hepatomas in B6C3F1 mice after high oral dosage [28). the biological significance of which being subject of present discussion [25,26]. ( Low degree of alkylation reported. 9+ Jt; T A i R&S 115641 2 H. M. Bolt. R. J. Lajb and J. G. FilsER This is consistent with Henschier's structural theory, and it lends biochemical support to the outcome of both mutagenicity [3, 7] and carcinogenicity (6, 13, 14,18-20,27,28,31] tests. Hence, there are different lines of support (metabolism and covalent binding, mutagenicity, carcinogenicity) to the view [33] that epoxides of halogenated ethylenes in fact must pos sess a certain degree of instability to effect a genotoxic 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 interpretable in this direction have now been accumulated. Metabolic rates andformation 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 haloethylenes, 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 (Ftg. 1). 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 preneoplastic nucJeoside-5'-tripho$phatase ("ATPase") deficient foci in rats exposed to these chemicals from the time of birth on; a detailed dis cussion of this methodology has been published [38, 39]. Table 2 contains metabolic data [36, 37] and data on formation of hepatic preneoplastic foci [25. 38-43] after exposure of \cung Wistar rats to halo ethylenes. A suitable endpoin* for such a comparison [39] is the percentage of ATPase deficient hepatocvtes after an exposure period of 10 weeks. The exposure concentrations of 2000 ppm ensured a satu ration of metabolizing enzymes (Vm conditions) [36]; only vinylidene chloride, because of its high acute toxicity, was applied at a concentration that produced a metabolic rate of about i Vm [37]. In accordance with Henschier's general rules (aide supra) trichloroethylene [38] as well as perchloroethylene [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 [38] and vinyli dene fluoride [41], This huge range is much reduced when the observed preneopiastic effect is related to the amount of haloethylene 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 vinyl chloride within their first week of life [44], C-C vinyl eWorld* H0 Xc-'c' ----- CHjCI--C 'a CH-CI-IS* ;onjugation[ a- -a c-c O Hientoro*thyl*ft* Ck .,01 C-C Cl" "Cl p*renioro*thyln* ,,H C-C H" Nflr vinyl bromidt "yV ^Cl civ^.vci Cl' ~ 'Cl CCI3_Cnh -- CCIj-- CHjOH \ ,0 ,0 CCU--C. --w ccu-c 3 'Cl 3 'OH A ,H - C--C ------- -* ' nr '8r Hn /H -C-C H" "F vinyl fluoride VyH H'C 'F ----- --- ? ,CI C-C H" 'Cl vinylidtn* chlond* C'ci CHjCI-C Cl #0 CHf^ "C'vO,H rgiutothion* conjugotion j ,F C-C. vinylidtn* fluoride A C--C 'f --- 7 Fig. 1. Initial steps of metabolism of halogenated ethylenes [2-5. 7. 11], < Haloethylene Vinyl fluoric Vinylidene f Vinyl chlori. Vinylidene c Trichloroet:, Perchloroe:*\ Vinyl bromc- Under *h: essential .:.r halooxiraccdeterminea equivalence the epoxic carcinoger.e for vinyl ch data in the i; idea of the different c. Ft m this ^ are available bromide ?_ physico-chr although the olism fvicyl [7]. But co-been public [46]; these fluorooxiri-i. than that cf; (B) When haloethyle*; ide and vi* compared intermediate tinctly low monohaloe are availab idene fluor* rane (the dr ably more the formeattempts o.; These twr tradict He: ; that instab oxiranes in. and carcir? yleMS. V re^He tc stroHurall wide rani, halogenatt Reactive metabolites and carcinogenicity of halogenated ethvlenes Table 2. Metabolic rates of haloeihvlenes and ATPase deficient preneoplastic foci observed after 10 weeks of exposure (Shr.day; 5 days/week) in newborn female Wistar rats Haloethylene Vinyl fluoride Vinylidene fluoride Vinvl chloride Vinylidene chloride Trichloroethviene Perchloroethvlene Vinyl bromide Exposure concentration (ppm) 2000 2000 2000 100 2000 2000 2000 Metabolic rate / pinole \ Ur-kg/ 7 36.37 1.1 36.37 110 36,37 50 [37] 210(36.37] 7 [36] 40(36. 37] Estimated amount metabolized during exposure (mmole/kg) 2.8 0.44 1 20 SJ 2.8 16 Preneoplastic foci after 10 weeks (9e of liver area) 0.04 40 0.0008 41 0.8 38 0.003 42 0 38 0 25,39] 0.07 43] Foci theoretically produced by 1 mole metabolites per kg b.tvt 14.3 1.81 18.2 0.15 0 0 4.38 Under the assumption [2-5] of Fig. 1 that the essential initial metabolic step is transformation to haiooxiranes, 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 the last column of Table 2 should give some idea of the comparative oncogenic effects of the different epoxides under realistic conditions in vivo. From this comparison, two features may be deduced: (A) The oncogenic effects of monohaloethylene metabolites decrease in the order: vinyl chloride > vinyl fluoride > vinyl bromide. No details are available on the metabolic pathways of vinyl bromide subsequent to epoxidanon 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 fluorooxirane and chlorooxirane [46]; these show that the 3-mcmbered ring of fluorooxirane has more tension and is less stable than that of chlorooxirane. (B) When the 1,1-dihaloethylenes and the monohaloethylenes (i.e., vinylidene fluoride vs vinyl fluor ide and vinylidene 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 monohaloethylenes. Whilst no experimental reports are available on a hypothetical [7] epoxide of vinvlideneHuoride, it is known [5] that 2,2-dichlorooxirane (the epoxide of vinylidene chloride) is consider ably more unstable than monochlorooxirane; hence, the former, by contrast to the latter, "resists all attempts of synthesis by conventional methods" [5]. These two points, at a first glance, seem to con tradict Henschler's structural rule [2-5] which says that instability of chlorooxirans (and possibly haio oxiranes 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, monochlorooxirane. 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. 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Res. 81. 11 (1981). t Printed n Ore. i Do Abst: gas t stud: prer Acute adr. (PCA) tc : re|Aion rotoxic ous svsti and 5-r. lowing depleiiois signih activity . able en; enzyme with irre serotone neurotox5( + )-PC.serotonif longer p does the in trypto iteration duration of Rl-)-' The pi PCA is reactive r * Suppm. T Author : should he : Currcn Biophysics acetyl chlf intrapentor..