Document rxw5v5pzrwjKQyE6LoZer9gM7

12/31/97 10:26 WKE W I V . MED CTR LIBRFERY 007 -AbstrPct Renal cell carcinomas IRCC) develop as a Key words Trichloroelbene .Renal cell cancer * consequence of somatic mutations of the von Hippel- VHL Tumour suppressor genes Lindau (V'HL) iumour supprtssor gene. Recent epide- miological studies show that high and prolonged 0 % ~ - - -- - ptional exposures lo irichloroethene (TRI) are assod- lltmwh at& with an increased imidence of RCC. Tumw fis- sues Trom 23 RCC patients with occupational histories Trichloroetbene (TRI) has been used for many decades of very high TRI exposure were analysed for somatic as a dtgreasing solvent in the metal and clectrpic in- mutations within the VHL gene.DNA msisdated from dusttries (8ruckner et al. 1989). Recently, high and pro- microdissected tumour cells, amplified by polymerase loaged occupational exposures to TRI have been chain reaction CPCR), and analysed in single strand reponed to cause mcrepsed incidence of renal cell car- conforn)ation polymorphism (SSCP} and sequcnciog. cinoma (RCC;Henschler el aJ. 1995; Vamvakas et al. RCC tissues ofa1123 TRI exposed personsanalysed rhus 19%). M m v e r , TRIexposed RCC patients show in- far showad aberrariom of the VHL gene, with 30% creased fmquency of abnormal urinary protein patterns having aberrationsin exon I, 44% in exon 2, and 26% in compared to unexposed RCC patients,indicating a TRIexon 3. By comparison to much lower reported ~ / N L associated tubular damage preceding or enhancing its mutationf'quencks of 33-55% in TRI-unexposed RCC nepbromrcinogcnic e h t (Mining et al. 1996). The ptients. these results indicate P specifically high mula- nepbrocarcinagenicityof TRI has been linked 10 specific tion frequency at the VHL gene in TRI-exposed RCC melrtbdites generated by the dutathione (GSH) depcnpatients; four of these aberrations have thus far k e n dant pathway. The initial GSH conjuple of TRI is confirmedas YHL mutationsby sequence analysis. This processed to dich)oroviuylcysreine (DOC), which is finding indicates the VHL gene being a susceptible and b ~ e dby renal tubular /%lyase to highly reactive specific target in TRI induced renal carcinogenesis. chlorinatedthiokelcnts. Such intermediates react readily Furthermore, the frqueal hwlvemfflt of exon 2 iden- with proteins and DNA (Vamvakasd al. 1993; Miiller tifies potential 'hot spots' Cor this carcinogen. in addi- et al. 1984). tion lo the ayailabfeepidemiologiarl studies h e resuhs A genetic hallmark dcommon RCC is the deletion are now further proof Tor human renal carcmogeniciry$ of gemtic material from ikshort ~ i r mof chromosome 3 induccd by high occupalional exposutes to TRI. (3p; Zbar et al. 1987). Based on these findin@, a gene responsible for m a l ca11ccc was hypothesized 10 be lo- cated within this chromooomal region. Recently. the I'HL gene has bqx isolated from that re@on by posi- tional cloning. and it could be demonstrated that this gene fullfilled the crireria ,ofa tumour suppressor gene {Lars et al. 1993): both homologous VHL alleles are mutated in she tumour tissue, and herflitary as well 8s spcrradic forms of KCC follow the Same two-hit muta- tional mechanism (Knub#m 1971). Thecodhg r*on of the VNL gene conwins 639 aucbtkks (nt) discnf?uted in three exons of 340 nl, 123 nt and 176 nt, respectrvdy. The idenlifice~ionof these seqwncff a l h dim1 in- tralpenic mutation analyses. Somatic VWL mutations .. 12/31/97 11%27 DUKE UNIU. MED CTR LIBw7RY ~ 008 333 have bcen identified by difY'eerent groups to occur in 33- 5%1 of sporadic RCC [Gnarra et al, 1994: Shuin ei al. 1994; Foster et a1 W 4 ; Whaky et af JW4),.About half of these known mutations rKm exon 2, which contributes ta less than 20% ofthe coding VHL sequence. Such disproportionate dislribution of somatic VHL mutations has been viewed as an indication of a preferential target rite forenvironmentalcarcioogttns. Inordertoelucidatea possible genotoxir:effect of TRI the VHL gene it is important to establish itsmulatiooal pattern in high-TKJ exposed RCC patients. This report indicates a specific role or TRI in human renal carcinogends. Twenty-three RCC tumurs were analysed for somatic mutations in the VHL gene. According to the semi- quantitative exposure assessmen1 by Vamvakas et at. ( 1997). nine patients were graded a TRI exposurehistory of level (+4) and 14 patients of leve( (++.t:Tabk 2). In comparison to VHL wild-type sequences. all patients showed an abnormal SSCP ptlttan in drher one of the VHL exons [Fig. 1). The SSCP varktions were located within exon 1 in 7/23 (30%) patients, within exon 1 in 101 23 (44%) patients and within exon 3 in 6/23(26%} pa- tients (Table 2). Currently we are plrforming further MmMaIBaadndlrl sequencing analyses d' these variations by SSCP. so far we have confirmed four S W P band shifts as J'HL mu- Twnty-three palknls with Ustobgkally vcrifisd RCC and with a specilk history of prolonged oecupaibl exposure io high concentralionsof TRIwen sludicd. Thesejmlienlswere obiaincd from two clinicel case-contd dudieb (mefinalized, cme ongoin@andfor wtrc pending 1 ~com1 pensation cases regarding an occupational dipasc due to TRI. An sxtcnsive rrview of ilx occupationcll history tations. Tumour number 1696 had a TC &Jetion of nucleotides 632 and 633, and tumor no. 1680 had a G deletion at nucleotide 637, both kxated within exon 2. The SSCPvariation of tumor no. 1677 mealed an 18-bp dektion at nucleotide 512 of exon I . The SSCP band was performed in dl paiionts. PamfRnsnibtddcd tumour iissuc of htse plrtknts was &aiixA from ~ h cr m p d v t cWiaca'Lpathow units which %re involved in s~11hgthe or$inal kistolog~cal diagnosis. shift of tuniour no. 1685 was due lo a nucleotide change from C lo T 81 position 81I in exon 3. The iiiutations in exon 1 and 2 were of frmeshifi type, rlie mutation in AB TRlsxposed RCC p i h i s had ken regularly hadliiig exon 3 was a inissense mutation. TRJ umlrr prcsntly unac~epubkconditions. R a m tempwitlures ra+ frequently between 30 and 50 4: in mall and poorly v&tilawd working arms. According to the sympt~msdescribed by the p t i e n ~C~a,nan~ra~ionofs TRI must haw exoetdcd M o l d tbc curiwit German oocupiional a;porurr lislir [MAK) of . mrSOppm. The patients reported that they often hd noticed a -W S ~ orU at work and dtm descrikxl fwwnliy recurring symptoms such as dizziness, headache, nausea and dmdlms, Many timcs a day the paricnts kR their wfkpl.oes for T ~ cCarcinogciiicity d TRI iS currently being mntroversially discusad. Chronic exposure io TKI led to an increased incidence of malignant liver, lung and kidney shot( periods of time, to get &csb air and to recover from thew turnurn in rats and mice (Maltoni et a]. 1988: NTP aclnc symptoms (see idso the desuiprians by Varnvakm et a]. 1996).The average time of exposun lo TRI of the plticnta studied was 21.8 years. Semiquantitative assessments of levcis of TRI EXpvwre are included in Table I . DNA of normd and tumour rksuc wus isohed urine @Amp 1990). In hliman epidemiology, four large cohort siudics on workers exposed to TRI showed no significanl increase of cancer morbidity or mortality (\Veiss 1996). However, m p s m concentralions mighl h a wbwn low tissue kit. Amplifiiliw of individutrl Yi3Lcxons was wit11 primer in these studies. and analysis of mortality instead of scte VHL 26/22 (exon I), 1M3 (exon 2) and YHlAhb (exon 3). !Sin& strand Oonronn~tioipi olymwpbn (SSCP) unalysis af thc pplymcrase cluin remian fPCRl products was in 8 and 1oo/o bumpdyiwxylilmidt gels. containing 0+09M TRIS-phosphate Mr. 0.02 M EDTA and 2%glycerol. Runniw conqdxd 0.02 M incidence might have inAnenced the ~esultsin cases of non-fatoll tumour~T. he observation of an increased incidence of RCC in workers with cxtremelr high and prolonged occupational exposure to TRI again raised . TRlSborateand 0.M M EDTA. Temperature during ekctropho- , nsis WES 4,15, or 25 "c. Sequencing of both minds of DNA was . cntried om by thc dye tmninatar method on a 373 DNA sequencer (Soquiserve]. Reisiled descriptions of the PCR. SSCP and se- the question of a carciaogmie e f k t of TRI in humans (I,fenschkret al. 199s). The rcsulls of this panicular study have been corroborated by a further case conuol quencing m e i h o d o l o ~mre given by G l m et el t 1996). study in the same seogrrphical area (Vamvakas et al. T.MC 1 Seniiquantitativc exposule asmsmcnt of persons oaxtpt~onallyexposed io iricMofocihent(TRl),accordin@ to Yrrnu4ias a a]. (19%). AI leilst one cri~siclllof each column bad 10 bc fulfilleJ in order to categorize the exposure lcvds Exposure kvd sr.<ting +++ +t DuraclonKrquency orexposure 2 5 yttrs andhi daily for at least 1 h Handling of TRl TRI temperature a60 "C and/or 200 1 Open TRI tubs A d m w CMdSizzi:ness. v e o , lwadmcheb ~3 'dweek, scwm prtnarcotic symplams 12131e7 10:29 D U E UNIU. PED CTR LIBRRRY ~ 009 334 Table 2 voii Hippel-Liiidau (VHLI muluticms iu renal ccll GIrCillolha (VCC) tissue or paticntr. related to age and TRI exposure dtiid 16.54 !bSS 1657 1Mi2 I663 1669 1670 1676 1677 1619 1660 Ja3 1684 1685 1687 f 689 1691 I 694 1695 1696 1697 1b98 1700 ~ ___ ~ $7 +++ 53 ++ 56 ++ 67 ++ 66 ++c 61 63 +t +t +i RJ 57 +++++ 65 ++ 73 ++ 55 +* 57 +++ 57 +++ 83 +++ 60 t + 59 74 38 tc++*c ++t 5_<-7< +++ ++ 61 +++ 56 ' +++ ~ __ 240 1 62 2 56 I 34 I 136 2 288 3 540 I I68 I %I 192 I 480 2 246 2 I32 2 360 3 390 1 4a I 251 3 420 3 1S? 3 58 2 312 3 300 I 460 3 - 1996f. In view of the strong and somewhat emotional tisiically signilkan1 correla~ionbelween TRI cxposure debate of t l h issue (Swam 1995; Bloemen and l'omen- and the 0oCurml)ce d R C C (Vamvakas et ai. 19%). the son 1995; Herischler et al. 1995b) further. independent present study investigates whclhcr the IWL gene is a lines of argumentation concerning the human renal specific molecular target for TRI.This is the first report carcinogenidty of TRI arc helpful. Therefhe. the in- to fink a known molecular genetic cause of RCC with troduction of new argumenis based on speciac molecu- exposure to a specific, and praclically important chem- lar biological features of TRI-induced renal cell tuniours ical toxicant. are of utmosi scientific and practical importance. We have identified aberrationsof the VHL gene in all A known causative inolPcular event in RCC are so- 23 RCC patients studied with high and long-tern oc- . matic VHL mutations (Gnarra et al. 1994). The VHL cupational exposure to TRi. Inilial sequencing resulls mutation patterns heretofore observed within exon 2, a confirmthesealterationsas being VHLmutationswith a frequent site of mutations in sporadk RCC. led to the potential loss or thane of the biologicalTunaion ofthe conclusion that emjmmnental carcinogensmight play a VHL protein. Thus, it js most likely that the high fre- major role in RCC pathogenesis. In Ihe light of a sta- quency of VHL eberralionsobserved here may be due to 'specific mutational evenls within the W f L gene. Thc mutational events afkciedexon I in W/i,.exon 1 in 44'50 N P 1655 1W 1694 andexon 3in26% ofthetumoursin TRI-exposedpersons, These resub arc tbe first indication of difkreatiation between TRl-cxposed and nai-TRI-expsed RCC pa- iknts with regard to YHL muration m e s and rrequen- cia. Whereas only 33-55'Ll of noll-TKI-exposed RCC showed VHLmutations(Giiarra el al. 1994; Shuin d a]. 1994; Foster et al 1994; Whaley et al 1994), 100% d the highly TRI-expbscd RCC pplients are afiected by such mutations, Also, despite the prcvious observations of frequent exon 2mutations in sporadic non-TR1-exposed RCC, these muialiolis accounted for only 24% of all tumours analysed (Gnarra et al. 1994). The high fre- quency (44%) ofexon 2 mutationsin TRI-exposed RCC patients, compared to the Imer mutation frequency in non-TRI-exposed patients, is apparently due to the po- tential of TRI to form chemicalintermediatescapable of interaaions with DNA. The responsible mechanism in- volves spBcific bioaaivation reactions. There are two competing pathways in rhe metabolism of TRI. The 12/31/97 10: 30 DUKE UNIV. MED CTR LIBRWY 010 335 P major Plrthwtiy involves biotrilnsformlriion by cylo- Knudsoa AG Jr 1197I) Mutation and cancer: slrctistical sfudp of chron.tr: P450. In lhc second pathway, TRl is conjuptcd by glutathione S-transferasesin a dose-dcpendani rcrinobtaslomr. Proc Na12 A d Sci USA 68:82-823 Lalif E Tory K. Gnarni J, Yao M,Dull FM,Orculr MI.. Stack- housc T.Kurmin 1. Mudi W. Grit L, Schmidt L. Zhou F. Li 1-1. tnnnncr. Glutathione conjugrtes derived from TRI are K c i MH.Chcn F. Glenn G ,Clioke P. Wrllhrr MM.Wcng Y . formed. especially folloiting high cxposure, and are Dum DSR. Dean M,Glavlre D,Kislinrds FM. Cmmy PA. further proccsscd into rciicrive intermediates with proti'ins and DNA (Miitlw et al. 1994). The present observations tire in accordance with this niechirnistic concept (Mllller et rl. 19941 and provide furthcr proof I'rrfusonJmith MA. Le Paslier D. Chmakov 1. Cohm D, Chintluh AC. Maher ER. LInehaii WM. Zbtlr B. Lcmin Mi ( I9931 Idcniiktion OF #hevon Hippet-tindru d i m e tumor wppritsor gmt. Science 160:1JI7.1320 Maltmi C. Lelmine G. Coni G . Perin0 CI (1988) Lunp-tmn for the human m a l mrci~~ogenico~fthyigh expsurcs to carcimgeniciry biotlssayr on trichloroethylene adimiiiiacrcdby TRI.In order to confirm fuurthcr the mutational e k t s of TRI 011 the I41L gene. we are currently exploring rbe hhalaibn IO Spr- Dawley rats and S w h and W 3 F l mice. Ann M Y W Sci 534: 316-342 M U l h M. h e r C. HenscW D. Dcknt W (1994) Strwtwcs of mutational patlcrns of the VHL gene in more detail, to DNA-adducs rormed Rom halokctmesa d halotlriok~csa: establish a specific 'genetic fingerpriat' of human RCC study IO identify possible adducts fanned from trichloro- and caused by TRI exposure. p.rchlorocrhenc through bioaaivalion by glutRlhione conjupiion. IARC Sci Pubi SET 115: 423428 KTP (1990) Cadnogemsir studies of trichaonrhykne (uitlioui epkhbrhydtin) ia F.WIM rats and B6C3F1 mice (gavage llehmcm srudkst. Technical Rcpofl243. Us Dcptlrrmeal d Health and Hurms Scrvias. National Indilutes d Htdllh trnd Human h-c Bloonen U.Toinenson J (19951Increased incideilce d'renal ccll i u m i s in R cohori of'csrdbtd aorkcrr exposed io tridtlorwtheile .-Comment. Arch Tosicol 70: 19-130 lkuckiier JV. Davis RD.Rhncal JN 419891 Mctsbolisni. loxicity. and carcinogcrlirit)- of trichloroeihenc. Cril Ret, 7oxicol 20: 31-40 Briining T. Gdka K. Makropoulus V. Boll HM (19%) Pivtxisienw of cbrw~ic:iubullr dnmagc iii pnses of rennl cell w m r afio. lolig aiid high nposure to iricbloroe(hcia.Arch Toxicol 70: 2S9--260 Foster K.h w s e A. van den &rgh F St. Hulrbcek MMF. Crossey PA, Richards FM. Cuirns P. Affnm NA. Frrguson-Smith MA. Buys ClKM. Malm ER (199.1) Soineric mulalions dthc t'on Hippel-Lindau discarc wtiw supprcswr SIX in non-iamilial clear ccll rend ulrcidoms. Hwn hld Genet 3: 21-2173 Glauic D, Ncanillan HPH. Wiltke C. Jachnig H. RMI ii. Meseli 0. Strticher T. hrusch F. Engdherdi D. Plalc K, H6ller H. Clm F. Zbw 3.Braucli I 3 f 1!W@iMutriimsin the IWL lurnor suppnsm p x alld associated lcsionr niih won Hippcl-Lindau discnje from Central Europe. I3um Geriei 98:171-280 Gmm JR, Tory K. Wen Y. Schmidt L. Wei M W ,ti H.Lafif F. Liu S. Glen 1:- Duh FM,Luhmsky 1. D a n DR. Floiance C. .Poz;taii R. Wahlier MM. Bmdcr NH. Grossma11 HB. b a u d H. Pomr S.books fD. Isaacs WB. Lwmn MI.Zbar B. Linelian WM (1994) Muiaiicms of ~hcf'HL tunlour suppressor genc in iriial Liirdnmfl. Nmm Gene1 7: $5-119 Henxhkr I). Vumvdtss S. Lanimcrt M. i)ck:)nt W. Kraus B. Thomas 8. Ulm K tl99stl) Increased -hcidencc of "I ccll turnom in a cdiori ofcardboard workers exposed 10 mchlorocrhmc. Arch Tcrxi~!al59: 291-299 Hcnschlcr D. Vtimvakas S. Lammeri M. I)ekeni W. Krrrus B. Thomas L), Uhn K 11995b) Incimeqnd incidcnct or renal cell Services. Berhesde. MD Shuin f .Kondo K. Toripoe S. Kishickt T. Kuboin Y . Hosaka M. Sagahismu Y, Kitamura H. LatY' F. Zbar 8. Ltmin MI. l-ao M (3994) Fregueni somatic mutlltions iind loss of Imcmrygosiry of the van HippetLindurl tumor suppressor enc in primary human renal cell eawinoina. Cancer Res 59: 3% S w c n GMH ( 1995) Increased incidcncc or renal ceil t m r s in a cohort of cardboard workers e~posPdto trkliloroellicne Cmmia. Airh Toxiool 70: 127-128 I'aimakas S. Dekmi W, Henschlcr D (1993) Nephroairanogei& ci1:- or hloRlkeicr and alkynes. In: Anders MW, r)ekzlni W. Henschkr D. Obedeithner N. Silbcraagcl S tats) R e d d i p siiion and nepitrotoxiciq of xenobiotics, Acadqic b s . San Diego. CA. pp 321-342 Vamvakas S. Bribing T. Thomas B, Lammert M. BatmUcr A. ldli HM. Henschkr D. Ulm K (1996) Renal d l cancer cor- relared \aid1 occuptional exposure to ~ricl~lorWhmSe.ee: Dcutsche Forschungsgetjwinsehit: Geaundhcilssch~dliFheAJbci\sss!oElc.-&eh H. 'Hcnschkr D (eds) T~khlnrrPhcll.22, Lkferung. 19%. VCH, Weinhcim. Kcis5 S S (19%) Cunm m relutioii to oaupationrt exposure to irichlorocihylenc. Oaap.Envlmn Mcd 53: 1-5 W'hak! JM. Naglich J. G e M 1.flsia YE,Lainiell JM.Gren JS Callins D. Neunlnnn HPH. Laidlaw 1, Li FI'. Kkin-Szanio AJP. hizittgcr B, Kfey N (1994) Germlinemutsliws in rbe ron Ilippcl-lintktu t u i m suppressor p c are similar to Eomuiic con Hippcl-llndau aberrations in spollrdic rmal ccl! emtinonu. Am J Hun, Genci 55: 1v92--1102 . Zbar 3. Brauch H. Talmadgc C,Linehnn M (1987) Loss of ~llclcs of loci on the sllort arm of cl~romosomc3 in renal rrll cnrci- t ~ m tN. a\ure 327 721-727 . tuinon in (I cnhori d cardboard workers exposed lo trichlo- mdhcnc - Reply. Arch Toxicvt x): 131- 133