There can be lit t le doub t that w e are en tering a new era in ou r understanding of the o rigin s of hum an cancer. U nfo rtunately f rom the po in t of view of the cancer ep idem io logy comm un ity, som e of the mo re recen t advances in the mo lecu lar b io logy of cancer (once fu lly assim ilated) w ill tend to m ake the talk of the up 2to2date cancer ep idem io logist a great deal less st raigh tfo rw ard than m any of u s had p reviou sly envisaged it to be, There m ay st ill be a few cancers that w ill p rove to resu lt f rom on ly a few dist inct ive types of m u tat ion in a relat ively sm allnum ber of genes, bu t I st rongly su spect that the great m ajo rity of hum an cancers that w e w ish to study w ill p rove to have their o rigin s in a comp lex set of DNA changes w ho se p recise m akeup w ill almo st certain ly differ f rom cancer to cancer, individual to individual, and popu lat ion to popu lat ion. I have no doub t that there w ill tu rn ou t to be a relat ively sm all set of genesw ho sem u tan t fo rm s are f requen t ly encoun tered in almo st any type of hum an cancer. Equally, how ever, I su spect that nonm u tan t fo rm s ofm any of the sam e genesw ill tu rn ou t to be impo rtan t in a sim ilarly w ide range of cancers, no t becau se of their w ild2type DNA sequences bu t becau se of a reduct ion (o r increase) in their ab ility to exp ress their encoded funct ion s resu lt ing f rom an ep igenet icmodif i2 cat ion of one fo rm o r ano ther (e. g. by CpG m ethylat ion). Mo re impo rtan t ly, perhap s, is the likelihood that a large m ajo rity of the commoner types of hum an cancer w ill p rove to have their o rigin s in a relat ively large num2 ber of separate genet ic and ep igenet ic changes to the ch romo som alDNA mo lecu les of cells of the o riginat ing t is2 sue. Som e of these changes m ay invo lve m u tato r (o r ep im u tato r) m u tat ion s; th is imp lies that no t all of the changes that lead to a cancer in a m u lt ip ly m u tan t (o r m u lt ip ly ep im u tan t) cellw ill arise independen t ly of each o ther. Sim ilarly, as a tumou r develop s and becom es sub ject to either genet ic o r ep igenet ic in stab ility, it m ayw ell acqu ire num erou s m u tat ion s (o r ep im u tat ion s) w ho se ro le in cau sat ion of the tumo r m ay seem obviou s at f irst sigh t (fo r funct ional reason s) bu t w ho se p resence in that part icu lar tumo r cell has no th ing w hat soever to do w ith the o rigin s of the tumo r. W ith th is background, it seem smo st un likely that the search fo r a single en2 vironm en tal cau se of a given tope of tumo rw ill be info rm at ive un less there are som e very unu sual circum stances w h ich help to en su re that it w ill be. Clearly there are casesw here“b roadscale”ep idem io logy has been ex t rem e2 ly successfu l, fo r examp le w here one studies the relat ion sh ip betw een a comp lex set of environm en tal variab les (cigaret te smok ing o r no t, nat ional dietary hab it s, degrees of sun ligh t expo su re, af latox in expo su re and related liver dam aging circum stances, etc. ) , bu t at temp t ing to ex tend the so rt s of app roaches that w ere successfu l in these cases to the iden t if icat ion of a relat ion sh ip betw een a single compound and a specif ic type of tumo r seem s to m e to be an ex t rem ely op t im ist ic endeavo r. Clearly m any of the m u tat ion s w h ich arise in m u lt ip ly m u tan t cancer cells m u st have their u lt im ate o rigin s in the ho st cell it self, so that m any of the m u tagen s invo lved are genu inely endogenou s to the ho st. Modifying spon taneou sm u tat ion f requenciesm ay therefo re be an impo rtan telem en t in carcinogenesis, and yet it m ay no t be discern ib le in any test system that is cu rren t ly in w idesp read u se. A lthough w e beve know n fo r m uch mo re than a decade that m any of the mo st common fo rm s of hum an cancer are m u lt im u tat ional in o rigin (and to th is w e m u st now add the po ssib ility of m u lt ip le ep im u tat ional changes) , w e seem to have been slow to act on the mo re obviou s imp licat ion s of th is fact. M y su sp icion is that in fu tu re studies of cancer in relat ion sh ip to the environm en t w e w ill need to change f rom a reduct ion ist ap2 p roach ( in w h ich one o r two variab les and a few genes are exam ined) to an app roach in w h ich comp lex ity is f reely acknow ledged and the in teract ion s of m u lt ip le system s are a m ajo r focu s of ou r effo rt s.

Mo st of the chem icals classif ied by the In ternat ionalA gency fo r Research on Cancer ( IARC) as hum an carcinogen s are m u tagen ic acro ss test system s, cf. www. epa. govög apdb < h t tp: ööwww. epa. govögapdb> and induce tumo rs at m u lt ip le sites in roden t species. They are therefo re readity detected in sho rt term test s fo r genet ic and related effect s (GRE) , in an im al carcinogenesis b ioassays and in hum an mon ito ring studies. Carcino2 gen s that are no t geno tox icm ay be studied u sing new tox icogenom ic app roaches asw ill be discu ssed. A Chem i2 cal Effect s in B io logical System s (CEBS) database is p lanned by the N at ional Cen ter fo r Tox icogenom ics to con2 tain info rm at ion on such compounds. The 1992 P ream b le to the IARCMonograph s p rovides exp licit gu idelines fo r including GRE in carcinogen icity evaluat ion s. In sub sequen tMonograph s GRE and o ther relevan t data have inf luenced the overall classif icat ion of at least 20 chem icals. Fo r examp le, the genet ic act ivity p rof ile of ethy2 lene ox ide (EO ) show s clear evidence of m u tagen icity in hum an s. EO con sisten t ly induces som at ic cell m u ta2 t ion s in roden t s in vivo, and gene m u tat ion, ch romo som al aberrat ion s, and SCEs in vit ro. There is suff icien t evidence of EO carcinogen icity in roden t s and lim ited evidence in hum an s. Th is, coup led w ith the compelling GRE that suppo rt a m u tagen icm echan ism of act ion, p romp ted the IARC to upgrade EO to a know n hum an car2 cinogen. IARC recen t ly reevaluated 1, 32bu tadiene (BD) as a p robab le hum an carcinogen. Bu tadiene is carcino2 gen ic in m ice and to a lesser degree in rat s. It is bo th m u tagen ic and clastogen ic in num erou s roden t studies, es2 pecially in m ice. Po sit ive resu lt s repo rted in th ree of eleven hum an ep idem io logical studies have indicated that BD may induce gene andöo r ch romo som alm u tat ion s in humans in vivo. We have review ed the GRE data for 1,32bu tadiene and fo r the o ther IARC p robab le and po ssib le hum an carcinogen sw ith hum an environm en tal exposu re. Th is assessm en t of the overall GRE fo r these agen t s, part icu larly tho sew ith po sit ive hum an in vivo data,undersco res the concern fo r adequate p ro tect ion of expo sed hum an s. Th is is an ab st ract of a p ropo sed p resentat ion and does no t necessarily ref lect EPA o r N IEHS po licy.

Werner’s syndrome (W S) and Bloom 's syndrome (BS) are rareau to som al recessive diseases in w h ich the featu re of premature aging and the elevated risk of neop lasia m ay be associated w ith genom ic in stab ility. To cha2 racterize the genom ic in stab ility ofW S and BS, B2lymphob lasto id cell lines (LCL s) f rom W Sand BS patients were cytogenet ically analyzed, comparing to tho sefrom healthy dono rs. A lthough allW S2LCL s exh ib ited sim i2 lar f requencies of spon taneou s m icronuclei (MN ) and sister ch rom at id exchange (SCE ) to the LCL s from healthy dono rs, BS2LCL s show ed ex t rao rdinarily h igh f requencies of MN as w ell as SCE. The cyto tox ic re2 spon se andMN induct ion to fou r chem icals, camp to thecin (CAM ) , etopo side (ETO ) , 42n it ro2qu ino line212ox de (4NQO ) , and m itom ycin C (MMC) w ere also exam ined. W S2LCL s did no t show any sign if ican t sen sit ivity in cyto tox icity andMN induct ion to all chem icals comparing to healthy dono r’s LCL s. BS2LCL s, on the o ther hand, tend to be cyto tox ic resistan t and sen sit ive toMN induct ion to 4NQO andMMC. Spect rum karyo typ ing (SKY) analysis revealed that almo stW S2and BS2LCL s generate“variegated t ran slocat ion mo saicism ”(V TM ) at h igh f requencies du ring cell cu ltu re. These f indings indicate that bo thW S and BS are ch romo som e un stab le syndrom es, bu t their characterist ics of genet ic in stab ility are to tally dist inct.

A n alkaline single cell gel elect ropho resis assay has been standardised by w h ich m u tagen sen sit ivity and DNA repair capacity (DRC) can be m easu red in cryop reserved peripheral b lood lymphocytes fo llow ing induct ion and repair of DNA dam age induced by b leom ycin. In an ongo ing case2con t ro l study, w e have app lied th is assay to Caucasian pat ien t s w ith non2sm all cell lung cancer (n= 160) and respect ive tumo r2f ree ho sp ital con t ro ls (n= 180). A f ter adju stm en t fo r age, gender and smok ing statu s, w e found a stat ist ically sign if ican t associat ion be2 tw een increased m u tagen sen sit ivity (OR = 4, 95%C I 2. 227. 4) , reduced DRC (OR = 2. 1, 95%C I 1. 124) and elevated risk fo r non2sm all cell lung cancer. Bo th m arkers em erged as independen t cancer risk facto rs. W e have also tested w hether cryop resevat ion inf luences m u tagen sen sit ivity and DRC. W hen f resh lymphocytes w ere compared w ith cryop reserved samp les, there w as no sign if ican t inf luence of cryop reservat ion t im e (1 to 371 days) neither on the amoun t of b leom ycin2induced dam age no r on the individualDRC as repeatedlym easu red in fou r b lood dono rs. W e conclude: ( i) in comparison to estab lished cytogenet ic test s, ou r new assay ism uch less t im e con sum ing, and m u tagen sen sit ivity and DRC can be assessed as independen t endpo in t s. ( ii) becau se it al2 low s sto rage of cryop reserved lymphocyte samp les, ou r assay is su itab le as a too l in mo lecu lar ep idem io logy. P ro spect ive studies to iden t ify h igh risk individuals and elucidat ion of the underlying m echan ism s fo r disease su scep t ib ility are now w arran ted.

Theab ility fo r DNA repair is an impo rtan t ho st facto r w h ich inf luences the individual su scep t ib ility to geno tox2 ic carcinogen expo su res. It has been show n in differen t case2con t ro l studies that DNA repair capacity (DRC) can be reduced in lung cancer pat ien t s. W e have u sed an alkaline com et assay to m easu re the cellu lar DRC in peri2pheral b lood lymphocytes of lung cancer pat ien t s and tumo r2f ree con t ro l sub ject s. A ddit ionally, fo r fu rther characterisat ion and in o rder to iden t ify DNA repair genes w h ich m igh t be respon sib le fo r tbe ob served impair2 m en t of DNA repair, w e developed a DNA array fo r exp ression analysis of hum an DNA repair genes: PCR f ragm en t s f rom I. M. A. G. E. clones of mo re than 60 know n genes, direct ly o r indirect ly invo lved in DNA re2 pair, have been p repared and u sed fo r array p reparat ion. So far, w e found a stat ist ically sign if ican t associat ion betw een reduced DRC and elevated risk fo r non2sm all cell lung cancer (P < 0. 0001). O u r cellu lar assay enab led u s to reliab ly iden t ify individuals w ith reduced DRC. Fu rthermo re, exp ression analyses w ere perfo rm ed w ith mRNA iso lated f rom individuals show ing either h igh o r low values of cellu lar DRC. The repo rted com et assay resu lt s w ill be discu ssed together w ith the individual differences in exp ression of DNA repair genes.

In the o ld indu st rial region of L a Louviere inW allon ia (sou thern part of Belgium ) 4 area’s can be dist ingu ished differing in sou rces of em ission s, popu lat ion den sity, and p resence of green zones. The cen t ral“L a Louviere2 Steelwo rk s”area is den sely popu lated and harbou rs b ig steelwo rk; the“Pow er p lan t2L andf ill”sem i2u rban area con tain s and o ld style landf ill, a coke oven and an o ld pow er p lan t fueled w ith coal. Betw een these two o ld in2 du st rial area’s lies themo re ru ral“In term ediate Green”area, w hereas the“Peripheral”area con tain s bo th ru ral and sem i2u rban zones bu t no impo rtan t sou rces of em ission s. Here w e p resen t the resu lt s of b iomon ito ring test s on healthy residen t s of these fou r area’s. Residen t s of the“L a Louvière2Steelwo rk s”(n = 58) and“Pow er p lan t2L andf ill”(n = 41) areas tend to have, compared to residen t s of the“peripheral area”(n = 65) , less p latelet sön l ( respect ively 248±51 & 245±61 vs 267±66) , mo re lymphocytesöLl (1, 915±5223 & 1, 928±5763 vs 1, 754±442) , mo re CD3 lymphocytesöLl (1, 344±4243 & 1, 344±4423 vs 1214±331, mo re CD8 lympho2 cytesöLl (661±2483 3 3 3 & 614±261 vs 545±193) , mo re CD3+ CD56+ lymphocytesöLl (121±104 (57) 3 3 & 125±99 (38) 3 3 3 vs 86±69) , a h igher comp lem en t C3 serum concen t rat ion and a h igher CEA serum concen t ration.

B iomon ito ring of the geno tox ic po ten t ial of occupat ional and environm en tal facto rs and their b io logical effect s in m an is essen t ial fo r en su ring p rim ary p reven t ion of hum an cancers and dam age of genet icm aterial. The group s of popu lat ion mon ito red in the Czech Repub lic fo r the occupat ional expo su re to clastogen ic geno tox in s comp rise b road spect rum of expo su re, the mo st of them invo lve comp lex m ix tu res of gases, vapo rs, and part icu lar m at2 ters. The increase of the ch romo som al aberrat ion f requency is con sidered as the signal of early adverse b io logi2 cal effect. Recen t resu lt s f rom Scandinavia, Italy and the Czech Repub lic show that an increased level of ch ro2 mo som e b reak s appears to be a relevan t b iom arker of fu tu re cancer risk. In the Czech Repub lic the cytogenet icanalysis has been in u se since 1974 fo rmon ito ring group s occupat ionally expo sed to geno tox ic carcinogen s. Du2 ring th is period m any thou sands of occupat ionally expo sed wo rkers have been exam ined f rom differen t p lan t and types of indu st ry. O ne of these, expo su re to PAH s, num ber of aberran t cells (AB. C. ) w as found betw een 2. 34% and 5. 07%. In popu lat ion group s occupat ionally expo sed to differen t vo lat ile chem ical compounds and their m etabo lites, w ith o r su spected carcinogen ic effect (ep ich lo rohydrin, vinylch lo ride, styrene, fo rm alde2 hyde, bu tadiene) the f requency of AB. C. w as ob served betw een 1. 05% and 4. 71% (con t ro l level 1. 81 % AB. C. ). Tbe ob tained data rep resen t a basis fo r quan t if icat ion of expo su re and fo r the app licat ion of p reven t ive m easu res (e. g. the change ofMAC).

Both expo su re and effect b iom arkersw ere m easu red in residen t s ofW ilrijk and Hoboken, indu st rial subu rb s of the b ig city of A n tw erp , and in residen t s of Peer, a ru ralm un icipality w ith in ten sive agricu ltu re (14, 622 inha2 b itan t s, 70 km east ofA n tw erp ). Person sw ith know n occupat ional expo su res, o rwo rk ing in a region w ith po l2 lu t ion levels clearly differen t f rom the area of residence o r comm u t ing over long distances, w ere excluded f rom the study. Here w e repo rt the HPRT varian t f requencies fo r 98 healthy non2smok ing wom en aged 50265 (46 f rom Peer, 29 f rom Hoboken and 23 f rom W ilrijk). The HPRT varian t f requency (V F) , determ ined u sing im2 m unocytochem ical stain ing w ith monoclonal an t i2B rdU , w as (m ean±s. d. ) 4. 8×10- 6±3. 5×10- 6 fo rW ilrijk, 13. 6×10- 6±31. 2×10- 6 fo r Hoboken and 19. 7×10- 6±49. 8×10- 6 fo r Peer. Fo r the to tal study popu lat ion, there w as no sign if ican t co rrelat ion (neither in a simp le linear regression no r in a stepw ise m u lt ip le regression w ith F to include= 1 and F to exclude= 0. 8) of V F w ith the fo llow ing personal o r life2style param eters: age, body m ass index, num ber of p regnancies, num ber of mon th s du ring w h ich b reast feeding w as given, con sump2 t ion ofm eat, of f ish, of baked o r grilledm eat o r f ish, of smoked o r salted m eat o r f ish, of o rgan icm eat o rm u s2 sels o r sh rimp s, of dairy p roduct s, daily in take of ethano l, of an im al fat, of calcium. A ncova analysis show ed that V F w as sign if ican t ly h igher in Peer than inW ilrijk af ter co rrect ion fo r age, fo rm er smok ing and level of ed2 ucat ion (P = 0. 0142).

In the period 199121999 w as carried ou t in the Czech Repub lic“Tep lice P rogram ”to evaluate among o thers the impact of air po llu t ion on genet icm aterials and p regnancy ou tcom es. DNA b inding act ivit ies of PM 10 w as stu2 died by an in v itro acellu lar assay coup led w ith 32P2po st labeling and em b ryo tox icity assay u sing Ch ick Em b ryo2 tox icity Screen ing Test (CHEST ). A ll analyses indicate that carcinogen ic PAH s are respon sib le fo r geno tox ic act ivity, con t ribu t ing 45% to 50% of allDNA adduct s induced by comp lex m ix tu res of o rgan ic compounds. In the P regnancy O u tcom e P ro ject w ere analyzed f rom 1994 th rough 1999 app rox. 8 500 p regnancies, u sing in t rau terine grow th retardat ion ( IU GR ) as ou tcom e m easu re. A sign if ican t ly increased risk of IU GR w as es2 tab lished fo r mo thers w ho w ere expo sed to PM 10 levels > 40 Lgöm 3 asw ell as to carcinogen ic PAH s > 15 ngö m 3 du ring the f irst mon th of gestat ion. Thu s, a p rim ary ro le of carcinogen ic PAN s in fetal grow th slow 2dow n w as hypo thesized. P lacen tal bu lky DNA adduct sw ere determ ined by 32P2po st labeling. DNA adduct sw ere relat2 ed to carcinogen ic PAH s expo su re. Resu lt s concern ing the impact of air po llu t ion on p regnancy ou tcom e show ed fo r the f irst t im e that air po llu t ion m ay inf iuence hum an rep roduct ive p rocesses, affect ing fetal grow th. The mo st impo rtan t f indings of Tep lice P rogram are: A ir po llu t ion m ay have an impact on p regnancy ou tcom e, DNA adduct s and som e geno types are sen sit ive b iom arkers of expo su re, PAH s are an impo rtan t sou rce of the geno tox ic and em b ryo tox ic act ivit ies of o rgan ic m ix tu res associated w ich u rban air part icles.

Exposure levels, noncancer effect s and su scep t ib ility facto rs can all be assessed in hum an s expo sed to envi2 ronm en tal carcinogen s. A dded to the vast arm am en tarium of t radit ional b iom arkers cu rren t ly availab le fo r these purpo ses are novel ones con stan t ly em erging f rom the rap idly develop ing areas of tox icogenom ics and p ro2 teom ics. The b iom arker arm am en tarium w ill be b rief ly review ed. T ran sit ional studies w ill even tually validate (o r fail to validate) each b iom arker fo r it s in tended app licat ion. O nce validated, b iom arkers of expo su re, effect and su scep t ib ilitym ay be u sed to relate studies in hum an popu lat ion s to tox ico logical evaluat ion s in test an im als fo r the pu rpo se of u sing the non2cancer endpo in t s in hum an s to bet ter def ine the hum an cancer risk. The N at ional Research Council’s paradigm fo r the induct ion of cancer by geno tox ic chem icals po rt rays the step s in the p rogression to m alignancy f rom ex ternal expo su re to f inal cancer p roduct ion. The in term ediate step s in the p rogression are in ternal do se, b io logically effect ive do se, early b io logical effect s and altered st ruc2 tu re and funct ion. These m ay all be m easu red by b iom arker respon ses, con st itu t ing the noncancer data that m ay be u sed fo r mode of act ion modeling and hum an cancer risk assessm en t. Comparison s of these endpo in betw een test an im als and hum an s, w ich cancer ou tcom es being assessed in the fo rm er, relate the endpo in t s cancer ou tcom es. A n examp le app licat ion of hum an b iom arker data to the cancer risk assessm en t p rocess w ill be con sidered u sing resu lt s of recen t studies of the chem ical agen t 1, 32bu tadiene (BD). BD is an impo rtan t, h igh2p roduct ion indu st rial chem ical. It is a po ten t carcinogen in m ice bu t m uch less so in rat s. Ep idem io logical studies in hum an wo rkers indicate an ino rease in leukem ia bu t the resu lt s are open to in terp retat ion. Fo r th is reason, BD w as classif ied by the In ternat ional A gency fo r Cancer Research ( IARC) as a“p robab le hum an carcinogen”(Class 2A ) bo th in 1992 and on reevaluat ion in 1999. The elect roph ilic m etabo lites of BD are unquest ionab ly geno tox ic in all species. BD’s roden t carcinogen i2 city, therefo re, has a geno tox ic mode of act ion. The elect roph ilic m etabo lites of BD are p roduced in v ivo w ith differen t eff iciencies in the differen t species, suggest ing that their rates of p roduct ion m ay determ ine the rela2 t ive carcinogen ic po tencies of BD. There is an abundance of non tumo r data, i. e. b iom arker respon ses, in m ice, rat s and hum an s that are in2 fo rm at ive as to the comparat ive m etabo lism and geno tox icity of BD. These w ill be review ed. Sub set s of these data have been ob tained under comparab le condit ion s in all th ree species, allow ing the direct comparison of re2 su lt s. These comparat ive resu lt s w ill be p resen ted in detail. In b rief, in th is sub set of studies, the mou se and rat expo su re BD concen t rat ion s ranged f rom 3 to 1, 250 ppm , w h ile the h ighest m ean hum an occupat ional expo2 su re w as～ 0. 8 ppm (eigh t2hou r t im e w eigh ted average). Desp ite these differences in ex ternal expo su re levels, there w as overlap in the concen t rat ion s of hemoglob in adduct s of BD m etabo lites betw een the hum an s and the reden t s at the low er expo su re levels. M easu rem en t s of u rinary concen t rat ion s of the differen t BD m etabo lites show ed clear differences in the detox if icat ion pathw ays u sed by the th ree species, w ith con jugat ion p redom inat2 ing in m ice and hydro lysis in hum an s. O f greatest relevance, the geno tox ic po tency of BD expo su re by inhala2 t ion w asm uch greater in m ice that in rat s, and no geno tox icity asm an ifest by increases in H PR T m u tat ion s in lymphocytesw ere seen in hum an s. These sub set comparison sw ill be con sidered in the con tex t of the overallBD b iom arker dataset. D irect ion s fo r fu tu re studies w ill be ou t lined and ten tat ive conclu sion s draw n.