Document 9JkVLdmEe9Y8obgz9v5bx6qD3
251,
Sinclair, P. R. and S. Cranlck. 1974, Uroporphyrin formation Induced by chlorinated hydrocarbons (lindane, polychlorinated biphenyls, tetrachlorodi.bcnr.o-p-dioxln) . Requirements for en dogenous Iron, protein synthesis and drug-mctaboliting activity. Biochemical and Biophysical Research Cotmtunlcations 61(1): 124-13).
HONS 200703
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BIOCHEMICAt AND CIOPHYSICAI RESEARCH COMMUNICATIONS
UROPORPHYRIN FORMATION Im-CKD BY CHLORINATED HYDROCAMiONS
(LINDANE, POLYCITI/TRIHATED HlKDtYU, TCTBACHUHCDIUZO-p- DIOXIN). *.' ri-
REQUIREMENTS FOR EMDOCEMDUf IMW. mn SYNTHESIS AND
DRUG-META00LIZINC ACTIVITY
rotor R. Sine loir end S. Crenlck
Rockefeller University Nov York, N.T. 10021
Received September 18,1974
Summery: Iho chlorlnotod hydrocarbon*, Undone (gemme-bexechlorocyclohexan*), polychlorinated biphenyl* end 2,3,7,6-tetrechlorodlbemo-p-dloxin, Induce clitck embryo liver cello In culture to rapidly occuaulote uroporphyrin 111. Thl* 1* in merkod centroot to the deleyed effect In *ulmel*i The Induction to dependent on protein tyntheil* (cyclohexlmldc Inhibited), endogenous lrou (chcletur* iii.lblted) end perhopo cytochrome P-430 (5KP-523A Inhibited). Evidence vas olmined fur on tinetehle Intenadiete thet le generated In the liver from chlorinated hdioc tibona which Inhibit* uroporphyrinogen docerboityleee thu* causing uroporphyrin to accumulate. The date luggaet that tho Intermediate Hy be a hyJror.yloti d derivative of tho chlorinated hydrocarbon.
Many Chemical*, Including berblturetee, eterold* end chlorinated hydrocarbon*
will Induce On lncreeee In porphyrin accumulation by chick embryo liver celt* In
eulturo (1.2). Thl* lncreeee in accumulation le precoded by an incrcaae In the
activity of Hie aniyme, delte-amlnolevullnlc acid eyntheieee-
Ihimane, other mammal* end eome bird* accumulate large enount* of uroporphyrin
In their llvere after treatment with chlorinated hydrocarbon* (3,4,S,6,7). However,
tho effect le delayed, varying from euverel day* In tho ceic of birds, to several
vooke fer maamal*. In thlt paper m will ehow thet th* cultured cell* respond
rapidly to tho chemical* to ipeelflcally accumulate uroporphyrin, Thl* accumulation
la dopendent on both protein eyntheela end endogenous Iron. Our result* mctfit
thet thcae celli may mctebollee the chlorinated hydrocarbone to *etivo moUi^liU*
which Inhibit the eniync, UROgen decerbowylaia
Heterial* end method*: The chlorinated hydrocarbone u*ed were: lindane (jar-a-
hexeehlorocyclohexnne) 99.51 fron Shell Company; Aroclor 1254, a mixture of
Abbreviations: URO, uroporphyrin; COl'KO, coproporpfayrln; rKOTO, pi uLuporpliyr I n ALA, delta-amlnolevullnic eeld.
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lOCHEMlC*t AMD ShOPHYSlCAl KESFARCH COMMUNICATIONS
ch'.orlnnted blphenyle predoslnently 4,5 and 6 ehlorlnea per molecule, from Monsanto; 2,4 ( 5, 2' ,4 * ,5* .licxachlorobiplicuyl from Ana lab*; and 2,3,7,B-tetr*chlorodlbenio-plioxln from Dow Chemical Company, All excapc the dioxin ware dlaaolved In ethanol. The dioxin un* dlaaolved In acetone. One Co two mlcrolltera of aolvant ware added per ml of eultura medium. Doafarrloxaolna was Oea feral from Clba and waa fraahly prepared in aallnc from the ampoule; the activity decreased vhen the powder la exposed to molct air but loss ao In i deaalcator at 4*. Rhodotorullc acid waa a gift from Or. H. Akcre of thla university, SKF-S25A (2-dlethylemlnoethyl 2,2- . dlphcnylvelerate HC1) wee e fife fro* Smith Kline end French laboratories. Plpcronyl butoxlde waa 96Z end vea e gift from FHC Corporation.
The composition and quality of porphyrin# extracted from cells or homoganotoa In IN perchloric acid (aqueous)rawthenol (1:1) were determined fluorometrleelly on the Hltochi-Perkln Elmer epeetrofluoromcter using the filters and phototube described previously (B). This procedure yields e 2-bended spectrum with peak* at about 605 end 660nm. The ratio* of the peek height* at thee* wavelengths permit rKOTO to be simply distinguished from ORO end/or COFRO. The actual Identifiertlom of URO waa confirmed by thin layer chromatography om elite* gal by the kerosene/ chloroform (2:6) eyotom. After determining the ratios of the peek heights, and the fluorescence yield ot 060nm of otendord eolutlon*. this method con bo used to easily glvo the 1 composition of FR0TO va. URO and/or COFRO and the quantity of porphyrins In mixtures. The method will be described In detail In a subsequent publication,
`to detenalno the laoewr compoaltlom of the URO'o they warm deeerboxylated to C0PR0 (9) end chromatographed on Eastman Chromegrama (silica gel) (10).
RESULTS The chlorinated hydrocarbons caue* the cells to accumulate uroporphyrin. Irma 1* Involved. Toble 1 shows that 2 ot the tested chlorinated hydrocarbons, lindane and 2,4,5,2',4',5'-haaeohloreblphmayl, oeuee the accumulation of URO, Thla 1* In contrast to ellyllsopropylacntmmldo and other inducer* dilch causa the accunilatlon of mainly FROl'O. Incubation of the cell* with any tested Inducer together with a chelator of Iron (o.g. doaferrloxemlne) caused the accumulation of PROTO rather than URO.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABU 1 Effect of chlorinated hydrocarbon* on
chick embryo liver calla in culture
Treatment
Porphyria* iii: pMlo/m* preCeS*
Type of porphyrin
None Dcefcrrloxamine (l.SsB) Allylieopropylecotemide (20C^ig/ml) AllyllBopropyUcitmldc + Dci!errloxnlni Lindane (3>i(t/ml) Lindane + Deaferrioxemina llexeehloroblphenyl (3ug/ml) Hcxftchlorobiphcnyl * Detferriox*lnc
12 16 900
1700 280
1100 90
2$0
PROTO PROTO 807, PROTO*
PROTO URO
PROTO URO PROTO
* and 201 URO + C0PR0 primary chick embryo liver call* were cultured a* described (11). The last change of medium was to the completely defined Ham-Fl2 medium containing insulin. Chemicals were added and porphyrin assayed in cells and medium after a further 16 hr incubation.
In other words, addition of the chelator removed the block in the biosynthetic chain
to that PROTO accumulated. The other chlorinated hydrocarbon* described In this
paper elan block porphyrin eyntheele at the URO etep. Of the chelators tested,
effective and non-toxic ones in appropriate concent rat lone were; desferrioxamine
(l.SaH), C*K& EDTA (equimolar Ca^+,
EDTA) (IOuLI), 8-hydrOXyquinol Inc sulfonate
(O.lntM), and rhodotorul ie acid (l.SmM). Anothar effect of the chelator of iron,independent of the effect on the kind
of porphyrin accumulated, la to increase the total porphyrin, i.c. to cause a potentiation. This 1* due to an Incrcaas in the total activity of the cnrwc, dclta-amlnolevulinlc acid synthetase (11). Active metabolism of .the cell* Is required for URO-orwAtetlon. In these cxpei l;<rn:'
'mu* :
\\N 61, No. 1, 1974
biochemical and biophysical research communications
TABLE II Requirements for URO aecumulatIon In tha presence
of different chlorinated hydrocarbons* In chick embryo liver cells in cultura
Ebtpt.
Addition
forphyrln
Required for URO accumulation
1 ALA** 2 ALA + Chlorinated HC 3 ALA * Chlorinated HC + Chelator 4 ALA + Chlorinated HC * Cycloheximlde 5 ALA t Chlorinated HC + SKP-525A
6 ALA + Chlorinated HC + Tlperonyl butoxlda
7 ALA * Chlorinated HC * Hama
PROTO URO PROTO PROTO PROTO
URO
Chlorinated HC
Endogenous Iron
Protein aynthaala
Cytochrome P-450 dependant matahe11 am
CytOChrMN f*450 dapandant mataboil am
Induction of ALA-aynthetaea
not required
* The Individual chlorinated hydrocarbon# in p&/l that vara uaad were: linden# (3); 2,4,5,2',4',5'-hexachlorobiphenyl (2); Aroclor 1254 (3); totrachlorodlbanao-
(0.03). **iThe concantrationa in pig/ml uaad were; for ALA, 25; for cyclohexlmlde, 0.1', for
ISKF-525A, 5; for piporonyl butoxlda, 5; for hem, 5. Cultura condition* and ^cheletor concentration* wara a* deacrlbad in legend to Tabla 1.
(Tchlo II) exogenous ALA was (Ivan ao that porphyrin production vaa not dependent on the activity of the collular ALA-aynthcteee. The total porphyrin fro* ALA whether URO or PROTO vaa about tha lana regardless of tha treatment. 1. The accumuletton of URO by tho call# vaa dapandant on protein aynthaala bacauaa cycloheximida (and emetine) blocked-VM formation (expt 4). 2. Endogenous iron ia raqulrad bacauaa In the prcfenco of chelators of iron, PHOTO, rathot than URO la formed (axpt 3). 3. Tho Inhibition by SKF-525A and plperonyl butoxlda (axpta 5,6) suggest* that tha compound# act via metabolic* poaaibly Involving cytochrome P-450 ilnce thaaa ara ctxupounda Ininvn to inhibit drug metahollaa (12). 4. the chlorinated hydrocarbons
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BIOCHEMICAI AND BlOPHYSICAl RESEARCH COMMUNICATIONS
have 2 e/fect*: they cause Induction of >nthoaU of AlA-3yTU)ieta*e, an effect vl:ic ! blocked by hone (11); and they causa URO accumulation from added AU, an effect idilrh la not blocked by heme (axpt 7), Evidence for a labile Intermediate generated l^^vjvo, dataefd In liver bontogenatea, and required for UBO formation. Liver bomotaaataa from oatroated chick anbeyoa when lncuhatad with AlA accuaulated PROTO (Table III), Addition of chlorinated hydrocarbons plot AU to the homogenate free untreated eebryoa did not effect the porphyrin produced. However, aa aaen In Table 111, when chick eabryoa were prevl< ouely treated with Aroclor 1254 and their Uvera were homopenlted end Incubated with AU, e ehort-llved component (or activity) appeared to be preaent In the homogenate which caused URO to accumulate. The short life of this component wee shown by preincubatlng the fresh liver homogenate for 1 hr, before addition of AU- This praIn cubation cauaad the loaa of tha ability of tha homogenate to fora URO froa AU, i.e.
TABLE III Evidence for labile compound that causae
URO accrmailatloa by liver homotenatae from chick embryos pretreated with Aroclor
Pretreetment of embryo
AROCLOR AROClOR
Pralneubatlon of homogenate before addition Of AU
4
Porphyrin accumulated after addition
of AU
Pteoro
PROTO
URO
PROTO
14-day chick ambryoe wars Injected with 15mg Aroclor 1254 in 0. 2al dimethyl aulfcodda. After 1.5 days homogenates ware prepared In 0.Z5M sucrose, 50aM trie chloride (pH 7.4) and pre incubated or not for lhr, 37*. Incubation volume wee 0.2ml containing 125yig wet weight of liver, AU (3wft> wee added to ell tubae after preincubation period and tubas were Incubated
for another lhr, 37*-
wlien AIA was added 1 hr. after preincubatf on, tlia porphyrin that wee formed wea PROTO. Thu* once the blocking effect cauaed by tho abort-lived component haa been removed, synthcaia to PROTO proceeda. The fact that PROTO la formed indicetea in addition that no damage of entywea from ALA to PROTO haa been cauaed by the chlorinated hydrocarbon or lte metabolitaa.
The liver honogenatea from control and Aroelor-treatcd embryoa both contained very little porphyrin and it waa PROTO. In the embryoa, excaee ALA ia evidently required to obaerve the effect of tho ahort-livad component in blocking et the URO atepKffcct of chlorinated phenola on URO production in liver honoienatay. Rata and mlea completely metabolite llndeno to a mixture of di-, tri-, tetre- and pente- chloroplicnola (13,14,15) and In part metabolite chlorinated blphenyle to the hydroxylatad derivative! (16,17), Tho poailblllty occurred to ua that a phenol metabolite might be
TABU IV Effect of phenola on URO production from AIA
by liver homogenetae
Addition
AU AIA + 2,4,6-trichlorophenol AIA + totrachlorophanol ALA penudilorophinol ALA * 3,4-dlnitrophanol AIA + pentectilorophemol chelator
2+ ALA 4 pntach|orophiu>l 4 fa AIA + pcntachlorophonol * dlphenyltmlao
Porphyrin aceumulated
PHOTO PHOTO 45* PHOTO*
umo URO URO URO URO
* and 55* URO Liver ho>i'Occnatc* from untreated embryoa vara prepared in logond to Table ill. Hicnola ucra InH added in l)il of ecatono, Choletore wero dccfcrrloxamlna (1.5mM); EDTA (lOmH); and a,e-dlpyridyl (0.5eiM). Oijdieuylamlno and Mn2* were imM,
129 an
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the actual blocking compound or related to it. Uo therefore tooted certain phenoli
(InN) pluo ALA (O.lSmM), added directly to liver homogenate* Iron untreated chick
embryos prepared aa deecrlbed In tlio legend to Table III, The 2,4, &-trlehler*phnl
caueod PHOTO accumulation. In contraat, the tatraehlorophenol eaueed 4S1 PR8TQ jS,
UHO forma!Ion. Pentachlorophenol and 2,4-dlnitrophenol caueod only URO accumulation Perliapa the clactron-u itlid rawing groupa such aa chloro- or nitre- on the aromatic rl;, function to atabllico some intermediate required for blocking at tba URO step,
rTr.vu. I / i ! -- id k-iso'1
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URO formation In the houwganato containing ALA and pentachlorophenol la aot effectod by the addition of a number of ehelators of Iron (l.SoH deaforrloxamlne,
i(t. 1. Siveiary of ac
IOmM KDTA, 0,SnM e,o-dlpyrldyl), Therofore Iron la not neceeaary for this psrtlcuU: reaction. This reaction la different from tho reaction that occurs In the cultured cells in Ml Jell the chlorinated hydrocarbon* require the presenco of iron to produce UHO (Table II, axpt 3). Attempt* to seek for the presence or e radical generated tlic clilorinertd phenol* wora uneucceaaful. Manganese ions and dlphenylamlne hsvs hen used ta block lipid poroeldatlon that ia generated by free radicals in rat liver uilcreaomoa (It). Wwn Un (1*R) or diphenylamine (ImM) were Incubated together vLth the ehlorophanols In a liver hemogenete they did net prevent URO accuuuletlon free ti
Homogenate* of embryonic heart treated directly with pontaehlorophanol accumulated Just aa much URO in tba presence of A1A a* did the liver homogenate*.
irip for the forirntio-ftutiollte toy be a h. vntaclilorophenol c*`j
llirec v-y nay b tj'.al.ly via met the
A) inhibition o' t: 1:11 1 ltoner woul if howogrootes of pi j-vver, the URA ite
found to b* mair
Tlii* *how* that the peatachlorophanol reaction la not specific to tha liver. Cyto chrome F-4S0 la probably vary low in heart homogenate. If cytochrome F-450 la lov It would ewggmet that cytochrome r-450 may not be involved in tha pentachlorophencl react lam to-block the tyntheala of heme at the URO atop.
PISCUSSIOW Figure 1 uaaarlree tha possible action* of tho chlorinated hydrocarbon* In tho blocking of porphyrin formation at tha URO step: 1. Thara la an induction of * nctohollr.lng system - this requires protein synthesis a Inca it is blocked by cycle* houlaidc. 2. The chlorinated hydrocarbon lo converted to a metabolite by a matabo* lining ivtu-o containing cytochrome P-450, since known Inhibitor* of cytochrome r-i> action (c.g. SKF-52SA) prevent URO formation. 3. Iren 1* raqulrod at soma rmactio*
: JS, ntho t*t
ok
to.iilsicnt with the
'ti the urine (20)B) There mny b
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Vol.6l.No 1,1974
8JOCHFMICAI AND 8IOPHYSICA1 RESEARCH COMMUNICATIONS PIS
IKIil'CTlON
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phenol)
) !);. 1. Suiiwary of action* of chlorinated hydrocarbon* to cauao URO accumulation.
nep for the formation of tho metabolite that cauaea URO aectwilatlon, and 4. Tha
rjiahollie nay bo a liydroxylated derivative of the chlorinated hydrocarbon* alnce
pntl irltlorophonol caused URO accumulation In the hoaoganata,
llircc ways nay bo conaldorod by which the chlorinated hydrocarbon* can act
probably via a metabolite (Rig. 1);
A) lull I bit Ion of UROgon III coaynthotaae. If thl* were the cat* then mainly
Hie URO 1 leomer would have accumulatod which la reported to occur when aupernatanta
of homogenatee of pig or human liver ere Incuhatad with AlA plua ferroua Iron (19).
Ilovevor, the URO Isomer ecctaeula tod by the cultured celle or the liver heawgeuatee
vc found to bo mefnly tho III feomar ee wo11 ee eoma I. Thoreforo inhibition of
rosjutlicUto ae a mechanism of URO formation may b* rajactad. nils oonolualon la
consist, enh with th* fact that rate traatad with hexachlorobonzono excreta URO III li> the urine (20).
B) Tlicro may be m.'Wery rapid direct oxidation of tho reduced UROgon III to ORO
111. In tho caao whero pontachlorophonol wae added to liver homogenates In the
presence of AIA,thc product formed wee UROgen ee Indicated by the fact that no
fluorescent porphyrin appeared,except *ft*r extraction and oxidation by Iodine or
In tho prcccnco of light. This rcault doe* not support tho Idee that the metabolite
of chlorinated hydrocarbon* generates a mechanism for rapid autoxldatlon of UROgM to URO.
c) Only a third possibility renaln*'. the Inhibition of the
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lOCHEMlCM AND SIOPHYSICAl RESEARCH COMMUNICATIONS
cnxyno, URAgon doearbeuylaae. We have not as rot directly demonstrated that tlit* cnrync 1* inhibited. Inhibition of this snsyme would bo oandlitout with I) the finding* of Teljasrd *1 (II) that th* imy* le aaapUtteljj^ef lclcnt in rot*
'.. " V'
treated for several weak* with both haxachlorobansataa and iron *nd 2) that lover
level* of the cnxync have boon found In rat* troetod with hexachlorobanrane alone (H. Tumlo and S. San Martin da Viola, personal coaaoinleatlon).
These finding* support the conclusion that the chlorinated hydrocarbons arc converted by liver natabullan to product* that Inhibit the anavoe, UROgn decarboxylase. Such product* have tentatively been identified the hydroxyl derivative* of the parent ca^ouad* which arc known natabolltc*. However no natobollte* hsva yat boon reported for the vary chemically Inert dioxin. It has therefore boon assumed that dlomlo act* directly. The rapid affect on chick enbryo liver cello Mon with dioxin suggest* that this nay bo o very good eystea for Investigating whether It ecto directly or vie some netabollt*.
Acknowlodtaeente: The author* wish to thank Ricardo Baldwin for technical assistance. The work waa supportad by grant USPHS; CM-04922.
manuals
1. crenlek, S, (1966) J- Kiel. Chao. 241, 1359-1375. I. Crenlek, tv and Kappa*, A. (19C7) J. Biel. Chew. 20, 4587-4593. 3. Oekner, H.K., end Schnld, >. (1961) Return 189, 499. 4. Do Msttele, F., Prior, B., end Unlagtoe, C. (1961) Mature 191, 363-34j. 5. Celdetoln, J.A., fllchaan, v., Bertpain, II., and Voa, J.c. (1973; Re*. Cc-Tv-n.
Chen. Pathol, Pharmacol. 919-92B. 4. Celdetoln, J.A.. Hleloaan,-P., end Jwe, kL (1974) Toxicol. Appl. rharnacol.
iZ, 437-448. 7. Strlk, J.J.T.W.A. (1973) Hnsyme 14 , 211-223 , 224-230. 8. Seise, 8., Crenlek, J.L., Crenlek, S., Kappas, A., and Icvarc, R. t). 0973)
Blachon. Mod, j, 135-148, 9. Rdnonaoo, P.X,, and Schwarts, 8. (1953) J. Biol. Chan. 205, fi0V609, 10. Falk, J.E. (1964) Porphyries and Metalloporphyrlne, pp. 191-153. r,l**vicr.
Amsterdam. 11. Sloclalr, P., and Crenlek, S. (1974) Ana. M.T. Acad. Bel. 1 pro**. 12. Andere, M.W. (1971) Ann. hav, Pharmacol. 11, 37-56. 13. Froal, J-J., and Ondwiek, R.V. (1973) Agr. Food Chon. JL *24-427. 14. Karapelly, J.C., Sehe, J.C., end hoe, T.W, (1973) Agr. :W Chco. 21. B1I-61P. 15. Kurlhere. N., end Rakajlma, H. (1974) Poetic. Bliahsn. l^fOlol. 4, 220-211. 16. Schulte, E., end Aekor, L. (1974) Naturaloenechoftee 11. 79-80.
MOMS 200712
V, Yoshimura, H, ,, Yamamoto, H., and Saekl, S. (1973) Cham. Fharm, Bui). 21. 2231-2236.
IB, Levin, V., Lu, A.Y.H., Jaeobion, H., Kuntzman, R,, Poyar, J. L., and HeCay, P.i. (1973) Arch. Bloehem. Rtophya, 15*. 862-852.
19. Kuahnar, J, F., Lee, C.R., and Naeht, $. (1972) J. Clin. Inveat. M, 1066-3051.
20. San Martin Do Vlale, L.C., Vlala, A.A., Naeht, S., and Crlneteln, H. (1970) Clin, Chin. Acta 28, 13-23.
21. Taljaard, J.J, F., Shonlay, B.C., Deppe, w,m. and Joubart, S.H, (1972) Brit. J.
Haematol. 23, S87-593.
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