Electrophilic halogens as potentially toxic metabolites of halogenated compounds

TIPS- February 1984 II

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dclx..nJclk'X ot the rcaL11on ~a~ ,,Iudlcd m gmmer d~ail (Fig 3A)~L For eumple. Ihe talc ~f membohmm of CCI, to an el~cUOlg~ic chlonne s p o c ~ and COCh increased as thc O~ concentration was decreased from I00 to 5% ~Filt. 3A): thi.~

I

Eloctrophilic halogens as potentially toxic metabolitos of halogonated compounds

v..u. Mmllar to the MIll:llh111 i1|v++t.,r~¢d dLltIn~ th." rcdu611s,c+dcc|l|orln,ltlOll of h+..xa-

Lance R. Poh! and Brace A. M ico Laboratory of Chemical Pharmacology, Natmnal H e ~ , Lung, and Blood Insutuw. ,'Vattmd l m m ~ o/ Health, Bet~exda, MD 20205, USA.

Ahmv drugs, msecticide.~, l)estici(h's, hed,i('i,h'.~, uud :,dlWm (-ounuu uhl,humcarh(m-halogcn hond.~. Ahhough it was once thought that ~u('h h+md.~ . e r e n.t broken appreciably m the body, it is now kmm'n that e.=vmanc dehulogenau+ms muv occur by several d~(ferenl me('hani.wm. Th(:w rea('ti, m.~ inch.h" uuch,cq~hlhc .~uhstitutions with ghttathione, oxidations, and redu('tion.~'. T'he.~e rea('ti(.t~ tre,luentl~ lead u) reactive intermediates, which m severed ca.+e+ appear to he revere.wide, at/ca.~t in part. for the toxicity seen after ex/)o.xure u) lhe pare+lt halogemued ((mq)oumt. Fhe ob/ect oJ'this review i~ to describe a re('endv oh~erred l~uh.'ay of metabolism of halogetutted ('ompolmd.s" u'hich int'cdi'es the fortHatlr)ll o f fief'el t'let'lrophlllt" halogen metal>olite.~. Identification of electrophilic halogen metabolites The fact that halogenated hydrocw~ns could be convened to electrophilic halogen metabolite,~ was demonstrated by incubating CCh with rat liver microsomes and 2,6.dimethylphenol (DMP) and detecting the fommtion of 4-chloro-2,~dimethvlphenol by gas chron)atography-n)a~,~ spectromet~' (Fig. I)". This finding indicated that DMP traPt~:d an electrophilic chlorine during the metabolisln of ('('h. OH

Medumism ofthe reactkm This idea has led us to postulate a pathway we call reductive.oxygenation (Fig. 2). The rtrst step of this reaction is the reductive-dechlonnation of CCI4 to trichloromcthyl nidical (CCb" ~, ~hich reads with Ch to form trichloromethyl~mxyl radical (CCI~DO.). This intermediate then decomposes to an eleclrophilic chlorine species and COCh. The most suggestive evidence for this mechanisnl ~ a s f o u n d xxhcn the oxXgcn

chhw,~.,thanc (('l,C-('(+Itl to pcatachhm+clhar~- ((+I,('-('II('I:! Fog 3(rJ m which pentachlot~hvl radical (CI+,C.-('Ch) is ~ l ~ v e d to be an inmmmediate'. Decreas. iP,g the 0 , concentration below apwoumalely 5%, however, did not further increase the rate of fcrmation of the eicclrophilk chh~rirm and COCG from CCI. (Fig .~.X) a~ ~a~, ~d~.'rxcd ~nth hcxaehkm~.-thalk."(Fig 3( "l but ll~ICad cau,~:d .0 decrease m the rate. as was seen during the oxidative demethylmion of ethyl morphine (Fig. 3B). At these low concenu'ations of Oz. it i~ bclicxcd thal the trapping of C(I,, by. (3= became rate delennmmg, and alternate reactions of ('Cb" bccan~: kmct0calh imlxmam. In this regard, it appears that only at low concenuations of O, does CCb(I) abstract a h y d r ~ e n atom from the media to produce CHCh; (2) become further reduced by cytochrome P-4.~) to form dichioromethyl carbene ( :CCh ) or ( 3 ) irreversibly reac~ wid, microsomal lipid~ The reductive-oxygenation mechanism is f'unber supported by the following oby~rrations: ( I ) CCh- has been spin-trapped with phenyl teniary-bu~Initrone dunng t ~ melal~di.~m or ( ' ( ' h h~ rat h~cr mlct,,+:.ore:,,*; (2 ~ pul.,~" P,lda+h'q'. ,.:ud,.", 11+dlcatc thai ~.'CI.~" rcacI~ rapidlx ~11h (): t,', Iorm CChOO-. which is appatendy unstable and decomposes to an unidentified electrophilic form of chlorine and ('(N.'h t g e t 2~: ~3~ ('Br('l.~, ~htch n~ rcducti~.ch dchalogenatcd more rapldl.x than t't'h. J~, al,~, metabolized to an elo:u'ophilic chlonne = and COCk (Ref I0) mon~ rapid!.~than is CCh; (4) in addition. CHCIs. v..~ich ~s nt~ reductively det-hlorinated, is n(~ melabol-

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Fig. I, Trapping of an electropMfi¢ chlorine membofite of C'(~+ by 2, o-dmwthyiplwnol ( DM P) in rat fiver mh,rosomes.

The reaction was found to be catalysed by cytochmme P-450 in rat liver microsomess'~ and occurred only in the presence of O=. Since CCk was also metabolized to phosgene (COCk) under these same conditionss,s it seemed possible that both the electrophilic chlorine and COCh might he formed by a similar mechanism,

/

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Fig. 2. Postldmedreducl~.o.t;v~natwn meclumismfi)r ttle me~Jbo~smofC(~l+ fo ~ ei~'troghdk" ¢ldormc specK,s and :20C1=by cytochrome P-4M).

62

T I P S -February1984

t00

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of CCI, to trichlommethyl hypochloritc volves a homolytic cleavage of the peroxyl (CChOCI) by a mechanism analogous to bond to produce COCh and an electhe oxidation of carbon-hydrogen bonds Irophilicchlorine radical (Fig. 4, pathway (R-H) to alcohols (R-OH) catalysed by A). The mechanism of this decomposition SO Cl,-'EleclroONlic Cl + COCI, cytochrome P-450. Although CCI~}CI has is analogous to that ascribed to the denot been synthesized, based upon the composition of a cytochrome /'-450 25 chemistry of analogues it would be heme--cumcoe hydrow~xide complex". expected to be a chlorinating agent which Alternatively, the beme..4richloromethylwould release COCh upon reacting with peroxyl complex may undergo intram 100 B . / / l r ' ~ - ~ : various nucleophilesLn. The fact that the molecular rearrangement to produce addition of cumene hydroperoxide or other C ( ~ h and an electmphilic chlodno-iron oxidizing agents to rat liver microsomes did hypochlorite complex (Fig. 4, pathway B). not support the metabolism of CCh to A similar intermediate has been postulated detectable levels of an ¢lectrophilic as the electrophih'c chlorinating agent of chlorine species or COCh, hut did support chloropegoxideseta. The fen'ic hypochlorite x< the oxidation of carbon-hydrogen bonds, complex may also react with water to pro. :E G~'-' • , , as well as the unique dependence of the dace the electrophilic chlorinating agent reaction on the concentration of O~ (Fig, hypochlomus acid (HOCI). 100' C. 3A)e'' indicated that the oxygenation of a Therefore, the electrophilic halogen ?5 carbon-chlorine honQof CCh did not occur produced during the decomposition of appreciably in rat livermicmsomes. trichloromethylperoxyl radical in rat liver 50 ~ Cl,C-CCI,--C,,C-CHC~, Conceivably, ~ichloromethylpemxyl microsomes might be a radical, a polar radical could produce an electmphilic species, or possibly both types of interchlorine species ar,d ~ , by any one of mediates. ,Q at least three cfifl'erent pathways. One pathway involve,; the reaction of two tri- Predktlon d metabolk reaetlvlty chloromethylperoxyl radicals to form a To date, only the tetrahalomethanes, % Oa tetraoxide interm~ate (CCIsO~Cls). This CCh, CBrCh, and CBr, have been shown, Fig. 3. Effect of oxygen concentration on (A) product would b= expected to decompose to be metabolized to electrophific halogen metabolism of CCI, m an. 'lectrophilicchlorine species" and COCi, (Ref. 6), (B) oxidative spontaneously to form O~, COCh and metabolitess.,. Thus, not all halogenated demethyla~on of ethyl morphine; and (C) an electrophilic chlorine radical". The compounds that are reductively-dereductive-dechlo6na~n of hexachloroetha~, to remaining two pathways involve an initial halogenated appear to be metabolized pentachloroethane¶by ratlivermicro.~omes. complexation of trichloromethylperoxyl appreciably by this pathway of metabolism. radical or its hydroperoxide derivative with One possible reason for this is that some ized to an electrophilic chlorine species~. ferric or ferrous cytochrome P-450, c~mpounds which are not metabolized to Alternative mechanisms for the metabol- analogous to the reaction,,; postulated for detectable levels of electmphilic halogens, ism of tet~alomethanes to an electroptfilic cumeue hydroperoxide with cytochrome such as halothane (CF~HCIBr), are only halogen Lntermedlate and C'(~h have been P-450 (Ref. 14). The heme-trichloro- reductively-dehalogenated at very low con. considered in detail, but they do not appear methylperoxyl complex is envisioned centratious of O~, presumably due to their to be major pathways for this reaction. For to decompose by a least two different inherently slow rates of reduction'.". Con. example, the possibility that the elec- mechanisms (Fig. 4). One process in- sequently, insufficient O, concentration tmphilic ~talogen is formed by an oxidation of halide ion, similar to the reactions cata+ 3 ar +4 0 +3 ¢x +4 lysed by chloroperoxidase or myeloperoxidase, w~ls excluded by showing that the + Cl" electrophilic chlorine metabolite of CCI, Cl "CI was derived exclusively from CCh and not 0 from chloride ion". The involvement of superoxide anion radical (O1--) in the reac0 CI tion was investigated because it is known to / C/...~ react rapidly &ith CCh, it can act as a Cl \el reducing agent, and it is normally produced during reactions catalysed by cytochrome /'-450 (Ref. 2). It does not appear to be involved in the metabolism of CCI, to an +3 or +4 0 +3 or +4 electrophilic chlorine species and COO,, however, because superoxide dismutase did not inhibit the formation of these pro. CI "C" CI ducts~7. Catalase also did not inhibit these reactions6'7, suggesting that hydrogen CI Cl peroxide was not involved in the metabolism of CCI4 to an electrophilic chlorine Cl CI species and COCh. Another pathway of metabolism consi- Fig.4. Postulatedpathways forthedecompositionof compleges of cytochrome P.450 and trichloromethyl. dered was one which involved oxygenation pemxyl radicalor ~ hydmperoxMe to an elec~rophilicform of cMorine and COCIL

,Q

T I P S - February 1984

available to trap the halocarbon radAlternatively, o t h e r c o m p o u n d s , such as h e x a c h l o r o e t h a n e (ChC--CCh). can be dechlorinaled in air but are still not metabolized to detectable levels of electrophilic halogen'. In the case of this compound, it appears that its radical i n l e r m e d i . ate, pentachk}methyl radical ( C I . K ' ~ ' C h is rapidly reduced by cytochrome P-450 to a carbanion (CCI.~CCh) which spontaneously dechlorinates to pr(aluce tetrachloroethene (CIK'---CCh)'. The finding that CIK:-CCh and penlachloroclhane (ChC-CHCh) were formed in a ratio of approximately 200:! supports this idea. Therefore, very little CIsC--~Ch seems to escape f r o m cytochrome P-450 to either abstract a hydrogen atom or react with molecular O= to form CI,~"-CHCh and

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tetrahalomethanes can he metabolized by the reductive-oxygenation mechanism to electrophilic halogens, the minimum struo. turai requirements will be the presence of an aliphatic carbon atom substituted with at least two halogen atoms. In the initial reductive-dehalogenation reaction to a halocarbon radical, one halogen will he lost as a halide ion (Fig. 2). This reaction will have to he able to occur at sufficient levels of O= for the halocarbon radical to he trap. ped as a peroxy derivative and will depend, at least in part, upon the reduction potential of the halogenated compound. The second halogen may he released as an electrophilic halogen upon decomposition of the halogenated peroxyl radical as earlier degTibcd. The type of carbonyl product produced during the reductive-oxygenation reaction should depend on the number of halogen atoms bonded to the carbon atom containing the peroxyl group (Fig. 5).

pholipids TM. Th~ fatty acid carbon radical could then react with O~ to produce a lipid hydmperoxide, which could cndergo decomposition. Similarly, halogen radicals may be initiators of lipid peroxidation. They may either react across double bonds of polyunsaturated fatty acids of phospholipids or abstract a hydrogen atom from a diallylic C-H bond from the fatty acid residue, in both cases, fatt~ acid carbon radicals would be produced which could react with O~ and lead to lipid peroxidation. If the electrophilic chlorine produced during reductiv¢-oxygenation was polar in character, like HOCI, it could react with p o r p h y r i n s , h e n ~ proteins, nuclco!ides and thiol containing molecules. These'types of interactions may bc responsible for the bactericidal effects of HOCI ( Rel. 2}. Carbonyl metabolites produced during reductive.oxygenation would ~so be expected to react with various tissue components and possibly be toxic. In this regard, recent studies indicate that the h e p a t o t o x i c i t y of CCI, m i g h t be parliallx caused by its metabolism to the hepatotoxin COCh (Ref. 20).

Potential toxicity of m e m b o E t e s produced by the reductive-oxygenation pathway e f m e t a b o l i s m Lipid p e r o x i d a t i o n produced by several halogenated compounds may he due, at least in part, to metabolites formed during reductiv¢-oxygenation. For instance, the fact that the o=aler of lipid peroxidation potency r' and metabolism by reductiv¢oxygenation~ correlat© in the series CBK?h > CCh >> CHCI, supports this idea. Moreov©r, it is known that CCl,.induced lipid petoxidafion m and its reductiv¢oxygenation"J (Fig. 3 A ) both occur at maximal rates at an oxygen concentration of approximately 5%. In this regard, CChOO. has been postulated to initiate lipid peroxidation by abstracting a hydrogen atom from a diallySc C-H bond of polyunsaturated fatty acids of phos-

Conclusion Compounds containing an aliphatic carbon atom substituted with at leaq t~,o halogen atoms may be metabolized in the body into reactive electrophilic halogens. It appears thai this reaction occurs, at least in pan, by a reductive-oxyg©nation mechanism. The first step of the reaction is catalysed by c ) t t x : h m m e P-4.q| and i n v o l ~ the reductive-dehalogenation of the halogenated compound to a halogenated carl,on radical. This intermediate reacts x~ith (): to form a halogenated pemxyl radical, which can decompose to an electrophili¢

halogen ~pcc-ic~ and a carl~m~l dcrl~ atl~ c !]!C h a l o g e n a t c d perox.xl radical, cicctrop h i l i c h a l o g e n , a n d c a r b o m l mctal~dltc,, l o m t c d d u r l n ~ the rcducti~c-ox.~gcnatlon reaction arc all l~tcnt,all.x tox,c m c L l h olites. Acknowledgements

The authors would like to thank Dr James R. Gillette for critically reviewing this manuscript.

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(Iq82) Btochem Pharmacol 11I. 301-~q~ '+ I'~¢~ ) I Xh.t .it I' I+ l ,ii I I% l:,t/+.ll I t+ .Ind [Ll~,, I H ,l'~'~') tt+,,, hc,u l+l,+t't:xs Res ('ommun 04. I I.¢~I-IIOU

I0 Pohl, L R . Bnmchflower, R s,' . }hghet. R I . Manta, J R, Nunn. D S. Monks. T J , G~,rp:+, I ~,k and llm.on I .'~ il~+~,ll D,.~: XI~++'I, D~sposttton O, .L14-339

II Mtoa. B. A and Pohl, L. R. gigS2) Hu~hem Bmphys. Res. Commun. 107.27-31 12 ~ s l u . A. T., Loew.G. H. Ma:o. B. g.. Branchflogcr. R X and Pohl. I R 4l,+s1+ J..'~t;! ('hi'Ill ,%cN" IIl~..L[~I.- .LIL~ 13 Howard. I. A., Bcnncn, j. E. and Brunton. G

~1981)Can J. Chem. 59, 225,L-2260 14 White,R. E. andCoon,M. J. (1980)Annu. Rev. Bkwhem 49, 315-356

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15 Lihby. R. D., T h o m a s , J. A . . Kai.~r, I. W Hager. I.. P (1982) J. Biol ('hem 5t)~l~-5t)37 16 Ahr, It J.. King. l J . NaM;linc,P)k,. W ttllnch, V 11t~82) Biochem t'harmacol 383-3th) 17 Xlt'q+d's | ) J . .]dll|V'.,..I I . ([,lX~'+,Oll {i .k Smuckler. E

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cGMP-depen d e nt protein phosphorylation in

in other manmlalian tissues including inte~ final brush borders, cerebellum and heart and m the connective nerves of .4ldy.~ia. The discover3 of these proteins has rehlforced the idea that cGMP carries out physiological roles which are distract 11"o!11 those mediated by cAMP.

the nervous system

I~rotein phosphor.vlation in lhe cerebellum in the past three years, imporl.nt advances have been made in the biochemical and immunocytochemical approach to cGMP function in nlammalian cerebeilmn. The cerebellum is unique among brain structures in that it is the only brain region in which cGMP levels equal or surpass those of cAMP. Depolarization of cerebeliar slices from mouse brain leads to a large and rapid increase in cGMP content suggesting an important relationship between cGMP and neural activity2. This functional relationship, combined with the relatively simple circuitry of the cerebellum and the availability of mutant mice defective in specific types of cerebellar neurons, has provided an appealing system for analysis of cGMP-dependent protein phosphorylation. Emphasis has been directed toward determining the cellular location of the cGMP-PK and characterizing specific sub,. strates for the enzyme. Knowledge cf the cellular location of the cGMP system is of obvious importance in assessing its possible functions and in designing strategie~ for pharmacological and physiological type experiments. Knowledge of the substrates is also important .qnce it is among them that one is likely to find the protein or proteins which actually carry out the ultimate functions of cGMP. The levels of cGMP-PK in the cerebellum are 10-20 times higher than in other major brain structures, such as cerebral cortex, hippocampus or medulla. This has been convincingly demonstrated by three independent methods: enzymatic assay of tissue extracts, photoaffinity labeling and by radioimmunoassay. Within the cerebel-

Dana W. Aswad Department o.f Pnchobud~gv. ~ ~mverxtty o f ('altfimua, irvine, t ;4 t~2 717, U S A .

Introduction ' Cyclic-GM,r' (cGMP) has been implicated as an'intracellular messenger in mediating the actions of several neurotransmitters and hormones in the nervous system; yet, to date, there is no cellular process for which the function of cGMP is well understood. This contrasts with the case of cAMP where a role in several physiological processes, most notably regulation of glycogen metabolism, is well established. Attempts to unveil the function of cGMP in neural systems have typically been of a physiological or pharmacological nature, e.g. to determine the effect of applying a neurotransmitter on cGMP levels in brain slices or to determine the effect of raising intracellular cGMP on a cellular property suck as membrane potential. More recently, a less direct approach, utihzing biochemical and immunocyto. chemical techniques, has been applied toward this goal. This latter approach is predicated on the basis that many, if not all, of the functions of cGMP are mediated by activation of a specific cGMP-dependent protein kinase (cGMP-PK) and that much can be deduced about ¢GMP function by stud'ying the physical properties and subcellular localization of the cGMP-PK and the proteins which it specifically phosphorylates. The purpose of this review is to sum.. marize some recent advances in this later approach to cGMP function and to indicate the directions that this research may take in the neat"future,

cGMP-dependent protein kinase The early history of cGMP research and the diversity of its possible regulator3, functions has been reviewed in detail La. A protein kinase specifically stimulated by low concentrations of cGMP was first di~ covered in lobster muscle and later in mammalian tissues (for comprehensive reviews of the discover3' and properties of cGMPPK see Refs 3-6). in mammals, this enzyme has a relatively restricted tissue distribution, with highest levels found in the cerebellum, lung and heart. The enzyme has been purified to homogeneity and extensively characterized, in contrast with the cAMP-dependent protein kinase, there appears to be only one isozyme of cGMPPK and it appears to be the same or nearly so in every tissue. Similarities between the cGMP-PK and tile cAMP-PK suggest that they may have evolved from a common ancestral gene. but there are important structural differences between these two classes of protein kinases. Whereas the cAMP-PK dissociates into catalytic (C) and regulatory (R) subunits upon binding cAMP [RaC2 4 cA --~ cA,Ra ¢- 2 C], the cGMP-PK does not dissociate upon binding cGMP [Ea + 2 cG ---. cG~.E~]. The discovery of cGMP-PK prompted a search for proteins whose phosphorylation was specifically stimulated by cGMP but not by cAMP. A group of such proteins was first found in the membrane fraction of mammalian smooth muscle. Specific substrates for cGMP-PK have since been found

1984. Elsexlet Soeno: PuNt~crs BV . Amsterdam ul~5 - 0147'84~E 110