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Id (hc,t. Io,,w. \ol. 21. "No. 2. pp 175 IS{I. IriS.~, t127k-{~915 ~43 l)211175-01bSli3.00 0 Printed in (treat lllitzun Pergamon Press Lid

NEGATIVE AMES TESTS OF FATTY METHYL DERIVED FROM HOMOLYSIS OF LINOLEIC ACID HYDROPEROXIDES*

H. W. GaRI)XIeR and C. G. ('R,~WI:ORI) Y,¢,'thcrn Rc{lional gt,.',t,tlrc h (',.'lllt'r. Aflricidtural Research 5ercice. (S l)cpartnient el .*lclriculturc#. Peoria. IL (H(,04 and J. T. MacGr~(;or |l'cstt,rn Re#toraH Rcwarch C('nter..'lHri('ultto'¢ll Re.~t'tll('h .~'ervice, t 'S l)t'parlt~l(,nl el ,tqric,lturc. Berkeh'.v. ('..1 94"~Io. I_'S.I

(Retcwed 9 .bow 19,~¢2}

Abstract Fixc isomeric epox~hydrox.',ene and epoxyoxoene fatt~ esters deri',ed from hemolytic de- composition of linoleic acid hydroperoxide were tested for mt, tagenicity by the "Ames" top-agar incor- poration method using s-9 mix derixed from livers of male rats pretrcated with Aroclor 1254. The cpoxidc fair.,, esters tested methyl trans-12.13-cpoxy-erythro-II-hydrox)-ciqtranq-9-octadcccnoate and methyl trwr,,- 12.13-cpox.',-threo- I 1-hydrox', -ci.sltransl-9-octadeccnc~ate leach composed o[ approximatcl.', SO",, cis-9-ene al~d 20"<> tron.~-t)-enel, methyl trtlns-12, lY-epox.',-tl-oxo-/r~in.s-II)-Ocladcceiloat¢, methyl trans- 12.13-epox)-9-h.x droxy-tran.> I O-octadecenoate and meth', I (-is-12.13-epoxy-9-oxo-tran.s- I O-octade- celloate had strtictural characteristics similar to certain potent nltllageils. Ilov,.evcr. these esters v, erc not mutagenic in Sal,rmelhl typhi,luriu,1 strains FAI00. IA98 or TAI537 at concentrations tip to 2000/ig.test plate. Under the same test conditions, the meth.',l of hydroperoxy linoleic acid. from which these ,.,.ere derl,.ed, was weakly, mutagenic m strata TAItR) and possibl', also m strain "FA'-)S.

Inl"rot)t (-eton such transformations are reactions of the guanine base pair with benzo[aJpyrene-7.8-dihydrodiol-9,10- Epoxidcs are produced as secondary products of lipid oxide (Jeffrey. Jcnnette. Blobstein ctal. 1976b), with oxidation, and some of these fatty epoxides are ,,icinal 7,12-dimcthylbenz[a ]anthracene-5.6-oxide (Jeffrey, to hydroxyl and oletinic groups. Such highly substi- Blobstein. Weinstem et al. 1976a) and with the puta- tuted [)arty cpoxides comp,ise an important group of tive 2,3-oxide of aflatoxin B~ arising from metabolic compounds found in mixtures as well as activation of aflatoxm B~ 0dssigmann. Croy, Nadzan in biological systems (Gardner. 1980). Epoxidcs as a et ul. 1977~. Such mutagenic epoxides are character- class are potential alkylating agents and some are ized by vicinal substituent groupIsl, which presumably' known to be mutagenic (Andersen. Kiel, Larsen & cause the epoxide to be electron deficient and thus Maxild. 1978: Greene. Friedman. Shcrrod & Salerno, rnore susceptible to nucleophilic attack. 1979: Ortiz de Montellano & Boparai. 1978: Thomp- In this comrmliaicalion we demonstrate that several son. ('oppinger. Piper et al. 1981 : Voogd. van dcr Stel epoxides derived from the decomposition of linoleic & Jacobs. 1981: Wade. Airy & Smsheimer. 1978- acid hydroperoxidc are not mutagenic an dctcrmincd Wade. Meyer & Hine. 1979). The possibility thal fatty by the method dcscribed by Ames. McCatm & Yama- epoxides may also be mutagenic has been suggeste~.t saki t19751. bv Mead (19,R0f Mulagenic epoxides arc often acti- vated in a wa,, that tacilitatcs reaction with a base- pair rcsiduc of I)NA. usually through nucleophilic EXpERI~IEN'I'.~I. attack on the cpoxidc. Among the known cxamples of Preparation of epo\ide fatty e,sters. Epoxide fatty esters were produced by decomposition of *Presented at a workshop on "'Lipid ()xidation. X"itamin l!. 13-hydroperoxy-cLs-9-tran,~- 11 -octadecadienoic acid Selenmna and Carcinogenesis" sponsored by the (linoleic acid hydroperoxide) followed by methyl es- National Cancer Institute and hekt at Rock,.'llle. MD. teritication of tile epoxide fatty acid formed. The on 15 16 September I?S0. mechanisms of formation of these compourids are dis- +lhe illenlioi1 of conlpall} ilaines or trade products does cussed m detail elsewhere {Gardner & Jursmic. 1981: not iinpl) that they are endorsed by the {.IS l)epart- most of Agriculture o'.er other firms or similar Ga.rdner & Kleiman, 1981 I. Two separate procedures products not mentioned. were u~d to produce the fatty ester epoxides and five 4bhretiations: I)MSO ~ I)imeth,,lsulplloxide; HPL(" ~- of the major products were isolated for mutagenieity high-prcsst, rc liquid chromatography: NMR = nuclear testing. magnetic resonance: TLC = thin-layer chromatog- In the first preparation procedure. 470 mg 13-hydro- raph~. peroxy-ci.s-9-trun.s-ll-octadecadienoic acid [99 + '!, 175 176 It. W. (3,XRt)>:I!R et al.

pt, re) was prepared by soya lipoxygenase (E(" posed of a mixture of 85". 13-hydroperoxy-9,11-octa- 1.13.11.121 oxidation of linoleic acid (Gardner. 19751 decadienoic acid and 15 °,, 9-hydroperoxy- 10.12-octa- for homol3tic decomposition with a cysteine-FcCl 3 decadienoic acid. the epoxide position of the esleritied catal3st (Gardner & Jursinic. 1981 ). The following iso- products reflected the same ratio. Thus compot,nd I[1 meric cpoxidc fatt', methyl esters with the epoxide consisted of 85". methyl trans-12.13-epoxy-9-oxo- located exclusively at thc 12,13-carbons (Fig. 1) werc tran.~-IO-octadecenoate and 15". methyl tran.s-9.10- isolated as dcscrihcd by Gardner & Kleiman (1981): epoxy- 13-oxo-trans-1 I-octadecenoate. These pos- meth31 trans-12.13-epoxy-erythro-11 -hydroxy-cis itional isomers, which difl~er only in the way the func- (transl-O-octadccenoate (I). methyl trans- 12,13-epoxy- tional groups are situated in relation to the fair.,, acid threo-I 1-h3.droxy-ci.s (tran.sJ-9-octadeccnoatc (IlL carbon chain, are so similar in their chemical and meth,, I tram,- 12.13-epoxy-9-oxo-trans- I 0-octadece- physical properties that they were not completely no~ltc (lII). meth31 tran.s-12.13-cpoxy-9-hydroxy- separable by chromatography. Therefore each lkltt~. trun.s-10-octadccenoate (IV) and methyl ciu-12.13- ester epoxide was isolated as an 85:15 mixtt, re. The epoxy-9-oxo-tran.s-10-octadecenoatc (V). Compounds esters prepared in this way were purified by column I and II were approximately 80°,, ci,s-9-ene and 2()"~, chromatography followed by HPLC. using the pro- trun~-9-enc. Because product IV was largely lost by cedure previously reported by Gardner & Crawford solvolysis, this compound was obtained by reduction (1981L Portions (15-20 rag) of epoxyhydroxyene and of zt portion of I by NaBH4 in methanol at 0 C. The epoxyoxoene esters were purilied by HPLC with 6 reduced ester was cxtracted from the reaction mixture and 4'~i, acetone in hexane, respectively. After HPLC into ('HCI 3 by addition of CHCI 3 and water to form separation, the isolates were washed according to the the mixture CHCI3 CH3OH-H20. 2:1:1 (by vol.). procedure of Folch, Lees & Sloane Stanley (1957]. Preparative TLC using hexane--, I:1 (v::v) was Compound IV was prepared as described above. used to purify the reduced ester. The purity of the Fractions that contained minor impurities, as assessed isolated esters was assessed by analytical TLC. and by analytical TLC, were purified further on prepara- the identity was confirmed by NMR and optical rota- tive TLC plates developed in hexane--ether, 3:2 (v:v). tor~ dispersion. Mutagenicity tests. Quantitative plate tests using With the second isolation procedure, larger quanti- the top agar overlay technique described by Ames et ties of epoxide esters were obtained from 1.9 g linoleic al. (19751 were used. Three hislidine-dependcnt acid hydroperoxide, as de~ribed by Gardner & mutants of Salmonella tjphimurium (TA 100, TA98 and Crawford (1981~. Since the hydroperoxide was corn- TA1537), obtained from Professor B. N. Ames. served as test strains. The S-9 microsomal fraction was pre- pared from Aroclor 1254-induced male rat liver as OH described by Ames et al. 11975) and was used at a level of 100/H S-9.ml of S-9 mix. Test materials (com- x pounds I-V above and the methyl ester of 13-hydro- peroxy-ci.s-9-trans-ll-octadecadienoic acid) were dis- 'OCH3 solved in dimethylsulphoxide (DMSO) and were OH added to top agar in a volume of 0.1 ml. Revertants obtained were confirmed b', subculturing on histidine- free media. Stability study. To confirm the stabilit,, o[' the epox- 0 0 ide fatty esters m DMSO. unused portions of the DMSO solutions used for the mutagenicity tests were ]]] ~OCH3 stored at -- 10 (" for 2 months and then extracted lot analysis. One part I)MS() solution was added to 20 parts CHCI3-CH3OH-He(), 2:l:1 (by ~o1.1 and the OH 0 CHCI 3 layer containing the test epoxide was washcd three times with water prior to analysis by TLC and ]~ ~OCH3 NMR. The epoxidc fatty esters recovered in tiffs way were found to be unchanged

OCH3 R E.';I." 1.1 S 9 ~ Thc epoxidc fatty esters were not mutagenic to the 0 0 histidine-dcpendent Salmonella typhimurium strains Fig. 1. Structures of fatty ester epoxides tested: (11 methyl used in the test (Table 1). The number of revertants tran.s- 12.13-cpox~-erythro- I 1-h ydrox y-cis( trans)-9-oetadece- per plate was not significantly greater than the con- noate : tII J methyl trans- 12.13-epoxy-threo- 1 l-hydroxy-cis- trol even when tile epoxide fatty esters were tested at ( trans)-9-octadeecnoatc : (I I I ) methyl trans- 12.13-epoxy-9- the 2000-/Lg level. Since there were no significant dif- oxo-traus- 10-octadccenoatc : (IV) methyl trans- 12,13-epoxy- ferences in the results regardless of the method used 9-hydroxy-trans-10-octadccenoate: (VI methyl cis-12.13- to obtain the fatty esters, test results obtained with cpoxy-9-oxo-tran~-10-octadcccnoate. Compounds 1 and 11 compounds from both of the isolation procedures are were each approximatel.~ 80", ci.s-9-ene and 20°,, trans.9- ene (thc lattcr structure is not shownl. Because the epox- included in Table 1. The pure positional isomers were ides v.erc derived from homol)tic decomposition of the tested up to the 1000-1~g.plate level, except for com- so',a lipox~genasc-produced 13-h',dropcroxidc of lmolcic pounds I and V which were assessed up to 5(X)itg. acid, the 13-carbon of tile epoxide was chiral as shown. plate. The material isolated frorn the large-seale prep- (p --:,i IL "6"~; " '~(,¥1 'It ")'~['I '6"S ) IF~II~'I :(L u)'~ IL "6"S LXIIVI :~Iaa.~lHm°(]S'l.. II" UlOl "6"S * Lt~.IVI "It .ILK: "his .:.t'~lVl 'It - .i :.t (,S ' XhVl Tit " I'19.1 "6-S x(,Vl it" ul Lq 'hS t (WIIVI :(.: .l 9~" 6-~ ntH~l 'l.~X.+[ :""(I:-; ]. :.o,,(1:,;I Hl~tll .~|otu (g l'~ll!J.~IpSI~IilIT% )I.[IPLII %~'I~;IJ.3ISI , +lqno(J (%l!itd lojlllO) II" J'~" [+'~IllLIIH~" JllTd I oJ10o3 tla1';)jr, lilt,all)2)111 tlll+J i ~;,3~ll3J3LJ;[+IOJltltO Oll"lld.~l llnlplXlpUl jo .3)lll+lJl)~ lll'J3",o %Ill b:Jll'dI'Ii'i'~ l*~Jllh)1ill J.lqttlflll:1((I ()] [l~flll.~HI I(II~ 111~llltlSl / )J:)q'l~)~lll to lip '"')(IS I :1111°(1~ 1) ;).)tlJl3|jl~}lll'~!/It]~l~ )kl?.~ 1 ~3"~.~J.~ll.~l~)lJlll):~ ",] Ill'ill ~J(~LI (~] JOJlUO~1L1311112'llO~ -~t(l i~lllp*a.a~x~.,~nllt~ :,Drul .'ISlDlslt .+I,~Ulg sdnOl~Ul p.~1~.~lspun()duJo~

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Lt=~l\,'J. ptm g6VJ. "(X)IV.I. Sll!l~JlSum!amU!lld.Q ~ll,)UouqD~; 8u!sn gp!m~ ,';111Uap!xoda jo ~,JOlSoI'~tllatu .]o s1~1 ';l!.~!tlO~)~ln['~"I olqRJ. - 17x It. W. GarDner eta/.

aration, which contained the mixed positional isomers, was used for the 2(X)0-/~g/plate tests as well as for the lower levels. +. ~-E ,.: "E At the higher dose levels, the growth of the back- = :,g,, ground lawn that subsists on the traces of histidme &. included in the media was inhibited m some cases, as + indicated in Tables 1 and 2. Methyl trans-12,13- cpox,~-9-hydroxy-tran~-10-octadecenoate and methyl 13-hydroperoxy-cis-9.trans-1 l-octadecadienoate were = I...... v- ?-o the most inhibitory. Of the five epoxy esters tested, the 12,13-epoxy-9-hydroxy-10-ene fatty ester (IV) was the most susceptible to solvolysis and NIH shift reac- tions of the epoxide (Gardner & Kleiman. 19791. .% _ In contrast to the results with the epoxy fatty esters. the hydroperoxide itself, methyl 13-hydropcroxy- ci~-9,trans-ll-octadecadienoate, was not only' toxic to Salmonella but also weakly mutagenic (Table 2t. Although the epoxide fatty esters in this study were readil3 solubilized by DMSO and did not appear to separate into globules in the overlay, wc tested the 0 esters after sonication with Tween 20 according to Yamaguchi & Yamashita (1979). The results we obtained after sonication (data not shown) were simi- lar to results using DMSO solubilization. On the other hand, methyl 13-hydropcroxy-cis-9.trans-ll- ocladecadienoate did form small globules within the ~r-,c agar overlay when DMSO solutions were used at the e% e-i ~ higher dose levels. However. sonication of the hydro- peroxide with Tween 20 did not yield reversion rates significantly different from those obtained by appli- cation of the sample dissolved in DMSO. &

,z-. -- % -'t , DIS( LSSION F-' Although epoxidcs are known alkylating agents and many are potent mutagens, the lack of mutagenic + = r- b.. effect with the epoxide fatty esters tested in this study is not inconsistent with previous published reports on structurally related compounds. These epoxy fatty esters possess two structural features reported to be associated with lack of mutagenicily. The first is ~*~ ,:: ~ t) -- .t 1.2-disubslitution. which generally results in markedly .= reduced mutagenicity compared with that of com- pounds with tile epoxy group in the (o position of an chain (Ivie, MacGregor & Hammock. 1980; Voogd et al. 1981: Wade et al. 1978). The epoxide rq fatty esters studied here belong to this class of hin- G~ dered epoxide. When mutagenicity of 1,2-disubsti- & tuted compounds has been reported, the substituent groups have generally been strongly electron-with- dra~sing. Secondl',, systematic studies of mutagenic- ally active epoxides of increasing chain length indi- cate that mutagenic activity' drops dramatically when the carbon chain exceeds 4-6 carbons (Thompson et al. 19gl: Voogd et aL 1981). presumably because of factors such as solubility and cell penetration. This it. ~ -- effect of chain length appears to affect genetic activity similarly both m Salmonella and in mammalian, in- cluding human, cells (Thompson et al. 1981). Our results, while not constituting a truly comprehensive A study of the potential genotoxicity of fatty epoxides, suggest that the genotoxic potential of these com- pounds may be minimal. The reactivity of compound IV (the 12,13-epox).-9-hbdroxy-10-ene fattv cster), as , ~ Z,~ ~ evidenced b3 its susceptibility' to solvolysis, presum- abl3 stems from the instability of the all vlic cpoxide. Non-mutagenicit? of fatty epoxidcs 179

Because (11 the susceptibility of this epoxide to nucleo- tire. Whether lipid oxidation products play a causal philic attack, it would probably be the most highly role in cancer initiation (or promotionl cannot be suspect as a tnutagen and. particularly in ,,'ie~ of the answered without much additional research. toxicity observed, further studies of this competed m other test systems would be of mtercst. .4cknowh,dqemcnt.s We are grateful to ('arol Wehr and Unlike the epoxide esters tested, methyl linoleate I)oris 1'. Frederick for technical assistance ~ith the Ames h.~droperoxide exhibited weak tnutagenicity under the test. and to Professor I'1. N. Amos, Uni,.crsity of ('alililrnia. conditions we u.~d. Lipid hydroperoxides are the Berkeley. (.'A. for providing the Salmonella nltltalllS. primary products of lipid oxidation, and ita turn the precursors of fatty epoxides. Recently. Yatnaguchi & RI'WI£REN('ES Yamashita 119801 also obtained a weak mutagenic re- Ames B. N.. McCann J. & 'famasaki E (19"751. Method> sponse ~ith isolated hydroperoxides of meth.,,I lin- for detecting carcinogens and mut~.ig¢iD, with the Salmo- oleatc and Iinolcnate ushlg thc method of Aines el a/. nella mlJmnlaliail-nlJcrosome 111tlt;.lgenlclt> test. .~llll~l- (1975 I. I t cannot be established with certaint) whether tion Re~ 31, 347. Andersen M.. Kicl P. Larsen II. & Maxild J. 1197~1. Mtlta- these results stemmed from the hydroperoxide itself gclli¢ actiOl/of aromatic epoxy resins. %'amr< l.oml. 276, or a decomposition product generated during the 391. course of incubatiorl with Salmonella. However two Cutler M+ G. & Schneider R. 119-/31. Mall\xmatLons pro- lines of evidence suggcstcd that the response was due duced m mice and rat', b', oxidized Imoleate. l-d (,~,mt,t. to the hydroperoxidc group rather than to secondary To'dcol. I I, 935. products of hydroperoxide decomposition: (al at simi- l)ix T. A. & Marnett L. J. (1981). Free radical epoxidation lar inutagenic response was obmr~ed with two other of 7.S-dila.',drox?,-7.S-dih.,,drobcnzo[ :~ ]p',renc b', hcnmtin hydropcroxides, tert-butylhydroperoxidc and cumene and polyunsaturated fatty acid ]Ddroperoxides../..Ira hydroperoxide, but not with peroxides, peracids and diem Soc. 103, 6744. F~,siglllalln J. M.. ('re', R. G.. Nadian A. M.. Iltlsb$ \V. I.. H2Oz {Yamaguchi & Yamashita. 19801: {bY the mttta- Jr. Reinhold V. N., Ili.ichi (i. & \llrOgall (.i.N. 11977i. genie ell;cot of autoxidized linolenic acid correlated SlrtiClUla] identification of the major I)NA addl.lCt with the pcroxidc value of the mixture (Yamaguchi & formed b) al]atoxitl ti, i ill vitro. I~t'ot . hath. -Iced 5( i Yamashita. 1979k It is. of course, possible that both [ ..S'..-I. ~4, 18711. these resuhs and our own could be due to decompo- Floyd R. ix.+. ~ootlg L M.. Walker R. N & .~ttlart M sition products generated during the course of the (1976i. l,ipid h?droperoxide activation of .\-h>drox>-\'- mutagenicity assay. On the other hand, Scheutv,'inkel- acetylaminofttiorenc \ Ja a free radical route. ('olation of,t pure isomer of hnolcic reported lhat autoxidized lmolcnic acid did not elicil acid II',droperoxide. l.ipid~ Ill, 24,S. increased rcxersion rates unless the fatty acid was (Jardner It. W. 119801. Lipid ell/\nits: lipases, lipox.vgen- sonicated with either detergent or protem ttowever, Lists. iHld "'h)droperoxidases". Ill ,tuto\idation in In,M we wcrc able to observe a weak response without the and Bi,Jogical Systems. Edited by '%1..'C;inlic& I%1 Karel. p 447. Plenum Press. Nov, "fork. use of special sohtbilization lechniqt, es. It is note- Gardner H. W. & ('rawlkn'd C. (5. 119811. Degradation of worlhv also that lmoleate hydroperoxides have been linoleic acid h)droperoxides b) a cystcine' I"c('l 3 catal,,,,t reported to haxe both toxic effects (Holman & Green- as a model Ik~r similar biochemical reactions Ill. A no\el berg, 1958t atld rcproductivc effects (Cutler & product, trony-I 2.13-epoxy-I I-oxo-lrems-9-octadcc,..'noic Schneider. 1973i m test animals. acid, fronl 13-1.(,~gl-IDdroperox)-cts-9.tran.s-I l-oetadecct- While the mutagcnicity of lipid hydropcroxides dienoic acid. IJio( hinl. tii(,,pJll~. .'l(t(I 6105, 126. may be duc to initiation of free radical chams, which Gardner H. W. & .lursinic P. A. (1981k Degradation of then cause Iormation of I)NA radicals IFukuzumi. linoleic acid h>droperoxlde~ b) a c>steine. I-e('l 3 catalyst 1978: Pictronigro. Scligman. Jones & Demopoulos, as a model lor Mlllilar biochemical reactll)llS 1. Stud} of 1976). the possibility that latly hydropcroxides may tix)gell requirement, catalyst and elTcct (11" pit. /llcreenmg of phen.,,Iglycidyl ether..%lutatmn Re', .w. Inasmuch as lipid hydroperoxides may cause car- 67, 9. cmogen actixation, this illustrates the necessity of Hohnan R. T. & (Jreenburg S. 1. (1958). A note on the considermg indirect pathways that may activate toxicitics el" methyl olcate peroxide and ethyl Imoleate secotldarv cellular conslituenls to a mutagenic deriva- peroxide. J..-Ira+ Oil (/we. 5"oc. 35, 707. ISO It. W. GARI)NI:P. et al. l,,ie G. W., MacGregor J. T. & Hammock B. I). (19~;0). ScheutwinkeI-Reich M., lngerowski G. & Stan H.-J. (1980). Mutagenicity of psoralen epoxides. Mutation Re~. 79, 73. Microbiological studies investigating mutagenicity of Jcffre3 A. M.. Blobstein S. 1t.. Weinstcin 1. B.. Beland F. A., deep frying fat fractions and some of their components. Harvc~ R. G., Kasai H. & Nakanishi K. (1976a). Lipids 15, 849. Structure of 7.12-dimcthylbenz[ u ]anthracene guanosine Thompson E. D., Coppinger W. J., Piper C. E., McCarroll adducts. Proc. mmr Acad. Sci. /_:.S.A. 73, 2311. N.. Oberly T. J. & Robinson l). (1981~. Mutagenicit.', of .letli'e.,. A. M., .lennette K. W.+ Blobstein S. H., Weinstein alkyl glycidyl in three short-term assays..~,4ut~mml 1. B.. Bcland I-'. A., Har~e.', R. G., Kasai H., Miura I. & Rex. 90, 213. Nakanishi K. (1976b1. 13cr~zoEa]pyrene-nucleic acid de- Voogd C. E., van der Stel J. J. & Jacobs J. J. J. A. A. (1981). rivative tound i~t city: Structure of a benzol a]pyrcne- The mutagenic action of aliphatic epoxides. Mutation tetrah'.drodiol epoxide guauosinc adduct..1. Am. ch~'m. Re.s. 89, 269. Soc. 98, 5,714. Wade D. R., Airy S. C. & Sinsheimcr J. E. (1978}. Mutagen- Mead J. F. 119~0). Membrane lipid pcroxidation and its icity of aliphatic epoxides. :~,lut~ttiml Rex. f4d, 217. prcvemion. J...tin. Oil ('hem. Soc. 57, 393. Wade M. J., Moyer J. W. & Hine C. H. (1979). 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