C hronic arsenic toxicity from ingestion of contaminated drinking water has been reported from many

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1 OPEN SUBJECT AREAS: RISK FACTORS PREDICTIVE MARKERS ENVIRONMENTAL MONITORING CANCER EPIDEMIOLOGY Reeived 17 Jnury 13 Aepted 7 June 13 Pulished July 13 Correspondene nd requests for mterils should e ddressed to A.K.G. (kgiri15@ yhoo.om; kgiri@ ii.res.in) or D.A.P. (dvid.poly@ mnhester..uk) * Current ddress: Deprtment of Physiology, Medil Siene Building, Room 7-1B, University of Alert, Edmonton, AB, T6G H7, Cnd { Current ddress: Shool of Environment & Life Sienes, University of Slford, Slford, UK High rseni in rie is ssoited with elevted genotoxi effets in humns Myukh Bnerjee 1 *, Nilnjn Bnerjee 1, Prith Bhtthrjee 1, Depriy Mondl {, Pul R. Lythgoe, Mrio Mrtínez 3, Jinxin Pn 3, Dvid A. Poly & Ashok K. Giri 1 1 Moleulr nd Humn Genetis Division, CSIR-Indin Institute of Chemil Biology,, Rj S. C. Mullik Rod, Kolkt 7 3, Indi, Shool of Erth, Atmospheri nd Environmentl Sienes nd Willimson Reserh Centre for Moleulr Environmentl Siene, University of Mnhester, M13 9PL, Mnhester, UK, 3 Proility nd Sttistis Group, Shool of Mthemtis, University of Mnhester, M13 9PL, Mnhester, UK. Arseni in drinking wter my use mjor deleterious helth impts inluding deth. Although rseni in rie hs reently een demonstrted to e potentil exposure route for humns, there hs een to dte no diret evidene for the impt of suh exposure on humn helth. Here we show for the first time, through ohort study in West Bengl, Indi, involving over humn sujets not otherwise signifintly exposed to rseni through drinking wter, elevted genotoxi effets, s mesured y mironulei (MN) in urothelil ells, ssoited with the stple onsumption of ooked rie with. mg/kg rseni. Further work is required to determine the ppliility to popultions with different dietry nd geneti hrteristis, ut with over 3 illion people in the world onsuming rie s stple food nd severl perent of this rie ontining suh elevted rseni onentrtions, this study rises onsiderle onerns over the thret to humn helth. C hroni rseni toxiity from ingestion of ontminted drinking wter hs een reported from mny ountries of the world nd is n environmentl prolem of olossl proportions 1 with wide rnge of deleterious helth impts, inluding hyperpigmenttion, kertosis, skin nd internl ners, nd vsulr diseses 1. More thn 3,,, people ross the world onsume rie s stple food 5. Arseni ontents of suh rie vries widely, with most reported onentrtions found in the rnge 9 mg/kg 5. Reently rie hs lso een identified s mjor exposure route 5 11, s evidened y oservtions of strong ssoition etween rie onsumption nd urinry rseni 1,13. Indeed it is often the most importnt humn exposure route where drinking wter rseni onentrtions re less thn 5 mg/l 8 1. The reltively high proportion of the more toxi inorgni rseni forms in rie 11 together with high rseni iovililities 1 nd ioessiilities 15 dd to the inresing plusile onern tht rseni in rie ould e helth thret to millions of people. Notwithstnding this, to our knowledge, there re to dte no studies tht demonstrte suh deleterious helth impts in humns onsuming high rseni rie in the sene of exposure through drinking wter. We therefore designed study to determine if ooked rie rseni ontent on its own is suffiient to give rise to genotoxi effets in humns. Results Demogrphi hrteristis of the study prtiipnts. The summry demogrphi hrteristis nd exposure sttus of the entire study popultion sed in rurl West Bengl, Indi, re shown in Tle 1. All the study groups (A F), seleted on the sis of eqully sped lssifition oundries with respet to ooked rie rseni, re similr with respet to their ge nd gender distriution, ody weight, nd lso their too usge. For ll groups, the men rseni ontent of drinking wter ws etween 3 nd 6 mg/l nd the men drinking wter intke etween.9 nd 3.8 L/dy. Comined with men ooked rie intkes of etween 5 nd 6 g/dy, this mens tht rseni exposure from drinking wter ontriuted no more thn % of totl dietry exposure for ny group, nd less thn 1% of totl dietry exposure for the study popultion tken s whole. Urinry rseni nd exposure from ooked rie. There is strong orreltion (r 5.81) etween grouped urinry rseni nd ooked rie rseni dt (Tle 1, Fig. 1) onfirming the overwhelming importne of rie s the mjor dietry exposure route in the study popultion. This reltionship ws lso found to vrying degrees of fits to ll of: mles, femles, too-users, non-too users, nd prtiipnts from eh of the 3 study res. SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 1

2 Tle 1 Demogrphi hrteristis nd rseni exposure sttus in the study popultion Group A Group B Group C Group D Group E Group F Totl study popultion Cooked Rie Arseni Rnge (mg/kg) #1.1 to.15 to. to.5 to.3 #15 # #5 #3 Numer of smples Gender Mle 9% % 5% % 5% 3% 7% Femle 51% 56% 5% 6% 5% 57% 53% Age (yers) Men (6SD)* (613.6) (61.5) (615.1) (61.) (61.8) (613.6) (613.) Too Use User 33% 6% 31% 9% 33% 35% 31% Non-User 67% 7% 69% 71% 67% 65% 69% Body Weight (kg) Men (6SD)* (68.) (67.7) (68.6) (66.3) (67.8) (69.6) (68.) Time t Home (Months/Yer) Men (6SD)* (6.8) (6.9) (6.8) (6.6) (6.8) (6.) (6.8) Arseni exposure Drinking wter rseni (mg/l) , 5.7, 5..1 Men (6SD)* (6.8) (63.1) (63.) (6.6) (63.) (63.) (63.1) Drinking wter intke (L/dy) ,d,f Men (6SD)* (61.) (61.1) (61.) (6.8) (61.) (6.8) (61.1) Cooked rie rseni (mg/kg) (Men 6 SD)* (6) (616) (61) (61) (615) (695) (61) Cooked rie intke (g/dy) Men (6SD)* (69) (69) (61) (68) (68) (611) (611) Urinry rseni (mg/l) ,, 96,, 5 Men (6SD)* (637) (6) (651) (67) (66) (681) (656) * One-wy ANOVA with modified Tukey-Krmer Multiple Comprisons Post Test. p,.5 ompred to Group A. p,.5 ompred to Group B. p,.5 ompred to Group C. d p,.5 ompred to Group D. f p,.5 ompred to Group F. Geneti dmge sttus s mesured y mironuleus ssy. For the whole ohort, MN rnged from.5 to.98, with medin of 1.91 nd n inter-qurtile rnge from 1.56 to.56. The whole ohort men MN ws (SD, n 5 17). On grouped sis, MN inresed monotonilly with men ooked rie rseni ontent from (SD, n 5 113) for the lowest ooked rie rseni group (A) up to (SD, n 5 37) for the highest ooked rie rseni group (F). Preliminry liner regression nlysis, using the ooked rie rseni groups s tegoril vriles nd with djustment for gender, ody weight, too usge nd drinking wter rseni onentrtion, showed tht groups with men Urinry Arseni (µg/kg) 1 y = x R² = Cooked Rie Arseni (mg/kg) Figure 1 Cross-plot of men urinry rseni vs men ooked rie rseni. The liner est-fit trendline is inditive only. Error rs represent 6 1 stndrd devition for eh prmeter for eh exposure group (A F). ooked rie rseni. mg/kg (D, E & F) eh showed signifintly higher (p,.1) mironulei frequenies thn the lowest exposure group (A), with the oeffiients for the predited inresed in MN for eh group reltive to the referene group (A) s follows: Group B:.5 (95% CI.15 to.5; p 5.631; Group C:.11 (95% CI.11 to.33; p 5.338); Group D:.66 (95% CI.35 to.96; p,.1); Group E:.83 (95% CI.5 to 1.15; p,.1) nd Group F: 1.38 (95% CI 1.9 to 1.68; p,.1). Further preliminry sttistil nlysis (One-wy ANOVA with Tukey- Krmer Multiple Pirwise Comprisons Test modified to ount for unequl group sizes nd vrine) shows tht groups with men ooked rie rseni. mg/kg (D, E & F) eh hve signifintly higher (p,.5) indution of geneti dmge ompred to eh of the groups with men ooked rie rseni,5 mg/kg (A, B & C), lthough reltively low p(.,.5) ws required y the w/s test invoked in order to fully omply with the requirement of this test for within group norml distriutions of MN. A more roust non-prmetri nlysis (Kruskl-Wllis test followed y Wiloxon Rnk Sum Test with ontinuity orretion) onfirmed the result tht ll the groups with men ooked rie rseni. mg/kg (D, E & F) showed signifintly higher (p,.5) mironulei frequenies thn the lower exposure groups (A, B & C) (Kruskl-Wllis Rnk Sum test (x ; df 5 5; p,.e-16); Wiloxon Rnk Sum tests for: groups A & D: W 5 951; p e-7; groups A & E: W ; p e-7; groups A&F: W ; p e-1; groups B & D: W 5 117; p 5.651e-5; groups B & E: W 5 86; p e-5; groups B & F: W ; p e-1; groups C & D: W 5 8.5; p 5.38; groups C & E: W 5 586; p 5 1.6e-5; groups C & F: W 5 5.5; p e-9) (Fig. ). No signifint differenes (p,.5) were found y either the modified Tukey-Krmer test or the more roust non-prmetri SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195

3 5 Mironulei Frequeny (MN/1 ells) A B C D E F (<= 1 (> 1 to <= 15 (> 15 to <= (> to <= 5 (> 5 to <= 3 (> 3 Figure Urothelil ell geneti dmge, s mesured y frequeny (per 1 ells) of indution of mironulei (MN), s funtion of totl rseni onentrtion () in onsumed ooked rie, grouped s indited. For this rurl West Bengl popultion onsuming rie s stple, high rseni in ooked rie is ssoited with elevted genotoxi effets. All groups with men As in ooked rie. mg/kg (D, E, F) hve men mironulei frequenies (MN/1 ells) signifintly higher (p,.5) thn those of the lower exposure groups (A, B, C). Numers in eh group s in Tle 1. Wiloxon Rnk Sum test etween the group men MN for ny pirs of groups with ooked rie rseni,5 mg/kg (A, B & C), ut euse of the smll smple size, we re unle to determine if this reflets tht there is no reltionship etween MN nd ooked rie rseni t these lower ooked rie rseni onentrtions or merely tht our study hd insuffiient power to detet suh reltionship. Similrly, the reltively smll numer of smples (n 5 5) for whih ooked rie rseni exeeded 6 mg/kg mens tht we re unle to determine whether or not the reltionship etween ooked rie rseni nd MN for our study ws more liner or su-liner for these very high onentrtions. Notwithstnding these limittions, the highest rie rseni ontent whih hs not een oserved to e unequivolly ssoited with signifintly inresed geneti dmge (Group C, Fig. 1) is mg/ kg, equivlent to 11 mg of rseni solely from rie soures eh dy nd, given the men ody weight of the study prtiipnts of 5.8 kg (Tle 1), equivlent to dosge of. mg As/kg-w/dy. Our results lerly demonstrte for this study popultion onsuming round 5 g of ooked rie per dy, tht ooked rie rseni ontent ove mg/kg is - on its own - suffiient to give rise to signifint mounts of geneti dmge, even when there is little exposure through drinking wter (Fig. ). Geneti dmge ssoition with high rseni rie is not onfounded here y other ftors. Age, gender, nd too-usge re often the mjor onfounding ftors in geneti toxiity study, ut the similr distriution of these ftors throughout the groups suggests tht it is unlikely for them to hve sustntilly onfounded the results. We note tht the sme positive reltionship etween mironulei frequeny nd rseni ontent of ooked rie is found in our study for oth men nd for women (Fig. 3), for oth toousers nd for too-non-users (Fig. 3), nd for eh of the 3 study res (Fig. 3). Questionnire-sed dt shows tht lmost ll of these individuls seldom trvelled outside their lol re nd lmost lwys used the sme wter soure, thus suggesting tht other soures of wter re not signifint onfounder (Tle 1). Comprle ody weight distriutions in eh study group t s proxy vrile tht shows tht the men rie intkes of the different study groups were lso omprle nd, s suh, the oserved differenes in geneti dmge sttus in groups with. mg/kg in their onsumed rie (Groups D, E, F) re, from preliminry inspetion, not likely due to differenes in the level of rie intke (Tle 1). Following this preliminry inspetion, in order to more roustly test whether or not key mesured ovrites, inluding ge, gender, too usge, odyweight (s proxy for ooked rie intke), drinking wter rseni, drinking wter intke nd study su-re, re signifint onfounders to the ssoition etween MN nd ooked rie rseni, stepwise (forwrds/kwrds) regression model ws onstruted utilising the dt for the whole study popultion (i.e. without onsidering groups A F). Distriution hrteristis, inluding the sling units, for these tegoril nd ontinuous ovrites re listed in Tle. The first step of this nlysis (Tle 3) reveled tht the only ovrites of signifine in prediting MN, expressed s (MN/1 ells) 1/, were, most signifintly, ooked rie rseni (p,.e-16) nd, to muh lower level of signifine, too usge (p 5.) with ll of the following ovrites tested not sttistilly signifint (p..5) onfounders, viz. ge (p 5.68), ody weight (p 5.11), drinking wter rseni (p 5.561), drinking wter intke (p 5.91), re (p 5.16). The finl model (Tle ) inludes liner nd qudrti ooked rie rseni terms, too usge nd gender s ovrites nd indites tht gender is not signifint onfounder (p 5.7), too usge is wek onfounder (p 5.78), wheres the overwhelming most SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 3

4 Mironulei Frequeny (MN/1 ells ) Mironulei Frequeny (MN/1 ells ) Mironulei Frequeny (MN/1 ells ) y=.9x+1.3 R²=.93 y=3.x+1.5 R² = Cooked Rie Arseni ( mg/kg) y = 5.1x R² =.91 y = 3.9x R² = Cooked Rie Arseni y = 8.87x +.83 R² =.88 (mg/kg) y =.17x + 1. R² =.9 1 y =.65x R² = Cooked Rie Arseni (mg/kg) Figure 3 Cross-plot of urothelil mironulei frequeny (MN/1 ells) nd rseni ontent in ooked rie for grouped dt. () mles (squres) nd femles (irles); () too-users (squres) nd toonon-users (irles); () groups from eh of the 3 study res, viz. Murshidd (squres), Ndi (irles) nd Est Midnpur (tringles). The liner est-fit trendlines re inditive only. Error rs represent 6 1 stndrd devition for eh prmeter for eh exposure group (A F). importnt ovrites in determining MN re the ooked rie rseni liner (p 5.5e-16) nd qudrti (p 5 1.3e-5) terms. The elevted toxiity of inorgni rseni speies ompred to orgni rseni moieties, suh s rsenoetine, rsenoholine, rsenolipids nd rsenosugrs, is well known 5,9, ut lthough we did not determine rseni speition in every ooked rie smple olleted, the perentge inorgni rseni (% i-as) ontent of lrge su-set of smples ws reltively uniform nd high (men %; SD; n 5 9) in greement with previous studies in the region 9,1,16 nd with only wek (r 5.) orreltion etween % i-as nd totl rseni, thus systemti vritions in the perentge of inorgni rseni in rie my lso e eliminted s signifint onfounding ftor in this study. Non-rie dietry soures of rseni, for exmple from vegetles, fruit nd sefood, my lso e redily eliminted s signifint onfounders in this study. Drinking wter intke of rseni hs lredy een shown to e low for the seleted study group s result of the study design. Hlder et l. 17 hve shown tht (i) the men ontriution to rseni intke in rurl West Bengl from vegetles is less thn. mg/kg-w/dy, orresponding to less thn % of totl dietry exposure; nd (ii) more importntly in reltion to the present study, the inter-qurtile rnge of suh intke is less thn.1 mg/kgw/dy. Rowhowdhury et l. 18 hs previously shown tht rie ontriutes over 9% of non-drinking wter dietry exposure to rseni in West Bengl. Lstly, historil exposure is nother potentil onfounding ftor for other ssys, ut the employment of MN ssy ensures tht the results we otined were only due to urrent exposure, sine MN is not n inheritle property of ell, rther eh ell quires MN during its short lifetime of eing exposed to rseni. Disussion Tht the ssoition oserved here etween mironulei frequeny in urothelil ells, nd rseni ontent in ooked rie is usl, is supported y () the strong positive orreltion of men urinry rseni with men ooked rie rseni ontent mongst the groups (Fig. 1) () over the rnge of vlues oserved, strong liner orreltion (r 5.96) exists etween men urothelil mironulei frequenies, nd men urinry rseni ontent mong the groups studied (Fig. ); () the oserved rte of inrese in mironulei frequeny per unit inrese in urinry rseni is rodly similr to tht previously oserved for similr popultions in West Bengl ut exposed to rseni lrgely through drinking wter insted of rie 19, ; (d) the previously inferred usl link etween rseni exposure from wter nd similr types of geneti dmge 19,1 nd (e) the met-nlysissed onlusion tht mironulei frequeny is meningful preditor of ner risk,3. We therefore elieve tht, for popultions, suh s the one studied here, tht my suffer from folte, niml protein, nd vegetle fire defiieny, ll of whih inrese the risk of toxi effets rising out of hroni rseni exposure nd onsuming rie s stple, eting ooked rie with greter thn mg/kg of rseni is unsfe. Although pprehensions regrding ill-effets of rie-derived rseni exposure vi the dietry route hve een in existene for quite some time, never hs this issue een ddressed experimentlly. Thus, ours is the first report whih provides diret evidene tht rie rseni ontent on its own is ssoited with demonstrle genotoxi effets, nd, in the se of the rurl West Bengl popultion studied here, t rseni onentrtions greter thn round mg/kg. This study provides sis for further similr ut lrger popultion sed studies, using mironulei frequeny nd/or other ytogeneti mrkers,3 to eluidte the puttive effets of other ftors suh s ge (inluding with respet to in utero, nd erly hildhood exposure) 5 7, gender, geneti onstitution 3,8 33 nd speifi dietry elements 3,,8 on the reltionship etween rseni exposure nd toxiity suh studies would e prtiulrly importnt to determine whether or not the genotoxi effets oserved in our study re representtive of impts in popultions, suh s in the Ameris, Europe, Afri nd elsewhere in Asi, tht re genetilly different nd often with etter nutritionl sttus. For our study popultion, mg/kg totl rseni ws equivlent to pproximtely 18 mg/kg inorgni rseni in rie nd to men SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195

5 Tle Whole group summry of tegoril nd ontinuous ovrites used in liner regression model of mironulei frequeny (MN/ 1 ells) in humn volunteers living in the rurl West Bengl study res nd onsuming rie s stple Ctegoril ovrites Covrite Ctegory 1 Numer Ctegory Numer Ctegory 3 Numer Sex Femle 3 Mle 19 Too User Yes 17 No 9 Are Very High 33 High 3 Low 161 Continuous ovrites Covrite Unit Min Q1 Medin (Men) Q3 Mx Cooked rie rseni mg/kg (17) 165 Drinking wter rseni mg/l d 1 3 () 7 1 Drinking wter intke L/dy (3.1) Age yer (38) 8 85 Bodyweight kg (51) Time t Home months/yer (11.8) n Very High (groundwter rseni hzrd) 5 Murshidd re; High (groundwter rseni hzrd) 5 Ndi re; Low (groundwter rseni hzrd) 5 Midnpore re. the following ovrites were not inluded in the model: urinry rseni (ovrint with ooking rie rseni), ooked rie intke (ovrint with odyweight). d elow 1 mg/l nlytil method detetion limit. dily intke of inorgni rseni of. mg/kg-w/dy. This intke vlue, ove whih we oserve genotoxi effets, (i) is mrginlly lower thn the PTWI (Provisionl Tolerle Weekly Intke) of.1 mg/kg-w/dy previously reommended y the WHO; nd (ii) lies in the region of the rnge of vlues (. 7. mg/kg-w/dy) reported y the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 3 s the inorgni rseni BMDL (Benhmrk Dose - Lower Confidene Limit) for.5% inresed inidene of lung ner: our study is therefore strong vindition of EFSA s (Europen Food Sfety Authority) onerns 9 nd JECFA s deision 3 to withdrw the inorgni rseni PTWI of.1 mg/kgw/dy 3. This study thus further highlights the inonsisteny of urrent ntionl nd interntionl regultion nd guidelines for rseni in drinking wter, nd rie 5,9,11,8,9,3 s well s ontriuting to the inresing evidene 35 tht, irrespetive of the exposure route, exposures to rseni muh lower thn the equivlent of L/dy 3 1 mg/l for 65 kg person my result in signifint genotoxi impts. With over 3 illion people worldwide onsuming rie s stple 5 nd with over 1% of tht rie in Bngldesh, Pkistn, nd Chin; over 5% of tht rie in Jpn, Itly, Frne, Spin; nd over 5% of tht rie in USA nd Frne estimted to hve rseni onentrtions exeeding mg/kg 5, even tking into ount vritions in the proportion of inorgni rseni in rie, the puli helth implitions re onsiderle nd wrrnt ontinued nd further onsidertion of regultory stndrds nd other instruments 5,11 to redue puli exposure to rseni vi this route. In the res investigted in this study, it is interesting to note tht whilst out % (1 of 56) of the smples olleted from highly groundwter-rseni exposed res showed ooked rie rseni ove mg/kg, only out % (3 of 161) of the smples olleted from reltively groundwter-rseni unexposed res showed the sme elevted rseni onentrtions. These oservtions point to irrigtion of rie pddy fields using high rseni groundwters s signifint use of high As-umultion in rie in West Bengl. Together with oserved yer-on-yer sesonlly-djusted seulr inreses of rseni in rie pddy soils in other similr prts of the Gnges-Brhmputr-Megn sin 36, this indites tht further review of irrigtion prties in these res is wrrnted. Suh review would need to refully ddress wider issues of how suh prties might negtively impt rop yields nd positively impt effiienies of utilistion of wter resoures. Despite the mgnitude of the prolem worldwide nd the re required to ensure tht hnges in rie irrigtion prties do not detrimentlly impt on rop yields, it is worth noting tht there re mny effetive nd potentilly effetive mngement strtegies for reduing rseni exposure from rie. Suitle ooking methods 37,38 nd ooking with low rseni wters n oth somewht redue rseni exposure. Mngement strtegies to redue rseni umultion in rie hve Tle 3 Summry of first stge of stepwise seletion of ovrites to inlude in model of mironulei frequeny, (MN/1 ells) 1/, for rurl West Bengl study group onsuming rie s stple. None of the ovrites re, ody weight, drinking wter rseni, ge or drinking wter intke re signifint onfounders for the ssoition etween MN nd ooked rie rseni Covrite Degrees of freedom Sum of Squres RSS AIC p (.x ) Sig. Code Model Are Body weight Drinking wter rseni Age Drinking wter input Too User * Cooked Rie Arseni ,.e-16 *** (MN) 1/, [Gender] 1 [Cooked Rie Arseni] 1 [Too User]. Signifine odes: ***.1 **.1 * Gender my e susequently eliminted (see Tle ) euse p(.x ). o SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 5

6 Tle Summry of model of mironulei frequeny, (MN/1 ells) 1/, for rurl West Bengl study group onsuming rie s stple Covrite Coeffiient Confidene Limits for Coeffiient (.5% & 97.5%) Stndrd Error t vlue p (. t ) Sig. Code (Interept) 1.151e1 1.85e e- 3.9,.E-16 *** 1.16e1 Gender (Mle) 1.113e e-.93e e- Cooked Rie Arseni 1.683e-3 1.9e e E-16 ***.7e-3 Too-User 6.31e- 6.61e e * 1.56e-1 (Cooked Rie Arseni) 6.99e-7 1.1e e E-5 *** 3.861e-7 Residul stndrd error on 1 degrees of freedom; Multiple-R 5.6; Adjusted R 5.189; F-sttisti on & 1 degrees of freedom, p-vlue,.e-16; Shpiro-Wilk normlity test for (MN) 1/ W ; p Signifine odes: ***.1 **.1 * Gender listed for interest only; it is not (p 5.73) signifint preditor of MN 1/. een summrised y Mehrg nd Zho 5 mongst others nd inlude promoting plque formtion nd the use of eroi ultivtion proesses 39. Utilising the reently disovered route of rie uptke of neutrl rsenous (III) id through quporins tht lso serve s hnnel for the uptke of silii id 3,,theuseofsuitleioville sili fertilizer supplements might lso e produtive strtegy where osts llow 5. Moreover, rseni onentrtions nd the proportion of inorgni rseni in rie vries widely, opening the opportunities to enourge the ultivtion of reltive low (inorgni) rseni vrieties 6 or the geneti modifition of rie 7,8 to redue rseni umultion in the grin 5.Solthough. mg/kg rie in rseni is ssoited with genotoxi effets in the studied ohort, whih onsumed rie s stple, there do exist vriety of strtegies wherey humn exposure nd susequent helth risks n e sustntilly redued. Methods Study site, prtiipnts nd smple olletion. Study res were seleted in rurl West Bengl euse: (i) the dietry ptterns re reltively homogeneous in rurl West Bengl reltive to the rest of Indi nd prtiulrly reltive to muh of the rest of the world 5 ; (ii) rie is the stple for the lol rurl popultion; (iii) there is overwhelming uniformity in the methods used to ook rie, with over 9% of the popultion using trditionl method involving repeted wshing nd then oiling in exess wter 9 (iv) lol rie ws known to exhiit wide rnge of rseni ontents 9,1 nd (v) rseni in lol rie is predominntly in the more toxi inorgni forms of rseni 9,1, thus minimising the requirement for rseni speition mesurements. Prtly in order to otin wide rnge of rie rseni onentrtions, we seleted study res in whih rie pddy field irrigtion wters hd ontrsting rseni ontents, viz. the very highly rseni ffeted distrit of Murshidd (Bhgwngol I lok), the highly rseni impted distrit Ndi (Chkdh Blok), nd the reltively low groundwter rseni distrit of Est Midnpur (Khejuri I lok). The seletion riteri for the study prtiipnts hve een desried in detil previously 1. For eh of the study res, the mjority of smples were olleted through informed medil mps orgnized y the CSIR-Indin Institute of Chemil Biology t pre-seleted dtes nd times in whih the villgers were requested to ttend irrespetive whether or not they exhiited ny rseni-ttriutle helth symptoms suh s rseni-speifi skin lesions. The lne of the smples were olleted through rndomised household surveys. Of those sreened, riteri for inlusion in the study were: (i) using rie s stple for t lest the lst 6 months; (ii) less thn 1 mg/l rseni in the household drinking wter; (iii) spending over 8% of wking hours in the sme re over the lst 6 months; nd (iv) greeing to prtiipte in the study y providing informed onsent. Study prtiipnts onsume the rie minly whih they grow in their own fields for t lest for 6 months in yer. For the rest of the time, they onsume lol mrketought rie, whih is grown in neighouring fields, thus mening tht they onsume lolly grown rie throughout the yer. Dt regrding soio-eonomi ftors, diet, too usge nd exposure history were ollted y n experiened non-physiin interviewer on the sis of questionnire. Eh prtiipnt provided us with informed onsent. From eh study prtiipnt, drinking wter, ooking wter, nd urine smples were olleted for nlysis (in this study, the drinking, nd ooking wter were the sme for eh prtiipnt nd hene oth referred to olletively heneforth s drinking wter). Initilly we sreened the drinking wter rseni onentrtion of over 6 individuls, from whih group of individuls met the sreening riteri, inluding #1 mg/l of rseni in their drinking wter. Cooked rie smples were susequently olleted from these individuls. Totl rseni nd rseni speition in ooked rie ws mesured y indutively oupled plsm mss spetrometry ICP-MS nd HPLC-ICP-MS fter extrtion following stndrdized protools 1. Bsed on the rseni ontent in ooked rie nd fter the removl of dt for 3 individuls for whom mis-oding errors were susequently found, the remining study prtiipnts (n 5 17) were divided into 6 exposure groups with eqully sped lssifition oundries s follows: Group A: #1 mg/kg; Group B:.1 mg/kg #15 mg/kg; Group C:.15 mg/kg # mg/kg; Group D:. mg/ kg #5 mg/kg; Group E:.5 mg/kg #3 mg/kg; nd, Group F:.3 mg/kg. Arseni estimtion. Anlysis for totl rseni in wter nd rie ws rried out t the Mnhester Anlytil Geohemistry Unit, University of Mnhester y ICP-MS (Agilent 75 Series ICP-MS). Totl rseni in lysed urine smples ws nlysed y ICP optil emission spetrometry (ICP-OES) (Perkin Elmer Optim 53DV). Urinry rseni onentrtions were not orreted for retinine ontent euse retinine hs een shown to e mrker for rseni methyltion effiieny nd hene possile onfounding ftor in our nlysis 5. Speition of rseni in rie ws determined y ICP-MS oupled with HPLC. Preservtion, preprtion, rie digestion, nd instrumentl nlysis methods were rodly s desried previously 9. Exposure ssessment. Totl dily intke of rseni for eh volunteer ws lulted y: As TDI 5 S As i IR i ; where As TDI is the totl dily intke; As i is the mesured rseni onentrtion in the susripted dietry item nd IR i the dily ingestion rte of tht item; only ooked rie, r, nd wter, w, were onsidered s importnt dietry items for rseni intke for the study popultion 9,1.IR w ws lulted from questionnire dt, speifilly s the produt of the volunteer s estimtes of the numer of drinks onsumed eh dy nd the volume of those drinks. Rie intke for eh of men nd women in rurl popultions in Indi hve previously een shown to e strong liner funtion of ody weight 9,51 nd so IR r ws lulted from mesured ody weight using previously pulished 9 reltionships: (IR r /kg/dy) (BW/kg) (for Mironulei Frequeny (MN/1 ells ) 8 6 y=.x+1.16 R²= Urinry Arseni (µg/kg) Figure Cross-plot of urothelil mironulei frequeny (MN/1 ells) nd urinry rseni ontent for grouped dt. The liner est-fit trendline is inditive only. Error rs represent 6 1 stndrd devition for eh prmeter for eh exposure group (A F). Dt from this study (dimonds): for whih rseni exposure is predominntly from ooked rie nd from Bsu et l. (squres) nd Ghosh et l. 19 (irles), oth for whih rseni exposure is predominntly from drinking wter. SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 6

7 mles) or.1651(bw/kg) (for femles) sed on the diet survey dt for West Bengl from the Ntionl Nutrition Monitoring Bureu (NNMB) 51. Mesurement of geneti dmge y mironuleus ssy. The effet of rie rseni ontent on ellulr dmge ws expeted to e sutle, nd hene, we hve used the sensitive mironuleus ssy (MN) in urothelil ells s mrker for hroni rseni toxiity through rie. Mironuleus ssy is n interntionlly well epted ytogeneti method 19 3,9,5 55 tht hs een extensively used for ner risk ssessment in humns 5 5. Indution of geneti dmge ws quntified y employing MN ssy in urothelil ells following well stndrdized protool 55. Briefly, ells were isolted y entrifugtion of urine, re-suspended in.9% NCl nd slides were prepred with 5 ml of ell suspension. The ells on the slide were fixed with methnol:eti id (351), stined with Giems nd sored under the mirosope following the riteri set down y Reli et l 55. At lest 1 urothelil ells were sored per slide nd slides sreened per individul. Sttistil nlyses. For preliminry ssessment of the dt, men ws used s the mesure of entrl tendeny nd one wy ANOVA with modified Tukey-Krmer Multiple Pirwise Comprisons Test, modified following the C-proedure of Dunnett 56, djusting for unequl group size nd vrine, ws pplied to test if the differenes in the entrl tendenies of different prmeters etween the different study groups (A F) were sttistilly signifint or not fter heking for withingroup normlity. Within-group normlity ws heked using w/s test ( 5.5) with the rtio of the group rnge to the group stndrd devition used s the test sttisti 57, orderline (,.5 to,.5) normlity tests were epted for groups for whih the smple size, n $ 3 (,.5 ws used for group D whih showed less normlity in the test for MN; nd for groups B & E for urinry rseni). Beuse of this non-norml pttern in one group of dt, non-prmetri tests were performed for heking nd ompring possile hnges in results nd onlusions. For this, Kruskl-Wllis test ( 5.5) followed y, for eh pir of groups, Wiloxon Rnk Sum test ( 5.5) with ontinuity orretion ws performed to mke omprisons etween the medins of the different study groups (A F). To undertke preliminry ssessment of the impt of rseni in ooked rie on mironulei, we performed liner regression nlysis with mesured mironulei frequeny (/1 ells) s the outome nd the rseni in ooked rie (mg/kg), either s tegoril (groups A to F) or seprtely s ontinuous vrile s the preditor, with djustment for gender, ody weight, too usge nd drinking wter rseni t survey. For liner regression modeling MN 1/ rther thn MN ws used s the outome with the im of omplying with the normlity ssumption of the model. Shpiro- Wilks tests ( 5.1) nd Q-Q plots were used for testing normlity in the dt (nd indited tht MN 1/ distriuted more losely thn MN to norml distriution). The liner regression model using the tegoril nd ontinuous vriles listed in Tle 3 s the ovrites ws derived following stepwise (forwrd-kwrd) model seletion proedure. For seleting ovrites, e 5.1 ws used s the riterion for entering vriles into the model nd o 5.15 s the riterion for eliminting them; for heking linerity, for eh ontinuous ovrite, the signifine of the oeffiients of the liner, qudrti nd ui terms were tested in stepwise proedure using the sme e nd o vlues s used in the previous step. Finlly, stepwise (forwrd-kwrd) seletion proedure for ll the pired intertion terms etween ovrites ws followed using e 5.5 nd o the reson for using lower signifine level thn in previous steps ws to ensure tht intertions terms would e inluded, dding to the omplexity of the model, only if they were strongly signifint. The modified Tukey-Krmer nlysis ws rried out using the DTK 58 pkge of R 59, the KrusklWllis test ws performed using the griole 6 pkge of R, the stepwise liner regression nlysis ws rried out using the lm nd step ommnds of R nd GrphPd InStt softwre (GrphPd Softwre, Sn Diego). All other lultions were performed using Exel (Mirosoft) (using pproprite dttype vlidtion). Ethil pprovl. Ethil pprovl for elements of this study were otined from the CSIR-Indin Institute of Chemil Biology nd the University of Mnhester Committee on the Ethis of Reserh on Humn Beings. 1. Smith, A. H., Lings, E. O. & Rhmn, M. Contmintion of drinking-wter y rseni in Bngldesh: puli helth emergeny. Bull. WHO 78, ().. NRC (Ntionl Reserh Counil) Arseni in Drinking Wter: 1 Updte. Wshington, DC, Ntionl Ademy Press (1). 3. Gi, H. et l. Utility of Reent Studies to Assess the Ntionl Reserh Counil 1 Estimtes of Cner risk from Ingested Arseni. Environ. Helth Perspet. 119, 8 9 (11).. Argos, M. et l. 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Helth effets of erly life exposure to rseni. Bsi & Clinil Phrmology & Toxiology 1, 11 (8). 7. Broekelheide, K. et l. Prediting Lter-Life Outomes of Erly-Life Exposures. Environ. Helth Perspet. 1, (1). 8. Kile, M. et l. The reltionship etween urinry rseni metolites, drinking wter, nd geneti polymorphisms in glutthione S-trnsferse M1 nd T1. Epidemiology 178, S19 15 (6). 9. EFSA. Pnel on Contminnts in the Food Chin (CONTAM). Sientifi opinion on rseni in food. EFSA J. 7, 1351 (9). 3. JECFA. -Evlution of Certin Contminnts in Food. 7 nd Report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Tehnil Report Series 959, 1 37 (11). 31. Engstrom, K. et l. Polymorphisms in Arseni(III) Methyltrnsferse (AS3MT) Predit Gene Expression of AS3MT s Well s Arseni Metolism. Environ. Helth Perspet. 119, (11). 3. Bnerjee, M. et l. Polymorphism in the ERCC odon 751 is ssoited with rseni-indued premlignnt hyperkertosis nd signifint hromosome errtions. Crinogenesis 8, (7). 33. Chen, C. J. et l. 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SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 7

8 39. Tripthi, R. D. et l. Arseni hzrds: strtegies for tolerne nd remedition y plnts. Trends Biotehnol. 5(), (7).. Tuli, R., Chkrrty, D., Trivedi, P. K. & Tripthi, R. D. Reent dvnes in rseni umultion nd metolism in rie. Mol. Breed. 6, (1). 1. Xu, X. Y., MGrth, S. P., Mehrg, A. A. & Zho, F. J. Growing rie eroilly mrkedly dereses rseni umultion. Env. Si. Tehnol., (8).. Tkhshi, Y. et l. Arseni ehvior in pddy fields during the yle of flooded nd non-flooded periods. Env. Si. Tehnol. 38, (). 3. M, J. F. et l. A silion trnsporter in rie. Nture, (6).. M, J. F. et l. Trnsporters of rsenite in rie nd their role in rseni umultion in rie grin. Pro. Nt. Ad. Si. 15, (8). 5. Li, R. Y. et l. Mitigtion of Arseni Aumultion in Rie with Wter Mngement nd Silion Fertiliztion. Env. Si. Tehnol. 3, (9). 6. Norton, G. J. et l. Identifition of Low Inorgni nd Totl Grin Arseni Rie Cultivrs from Bngldesh. Env. Si. Tehnol. 3, (9). 7. Dun, G. L. et l. Expressing SACR3 in Rie Enhned Arsenite Efflux nd Redued Arseni Aumultion in Rie Grins. Plnt Cell Physiol. 53, (1). 8. Meng, X. Y. et l. Arseni iotrnsformtion nd voltiliztion in trnsgeni rie. New Phytologist 191, 9 56 (11). 9. Sengupt, M. K. et l. Arseni urden of ooked rie: Trditionl nd modern methods. Food Chem. Toxiol., (6). 5. Bsu, A. et l. Cretinine, Diet, Mironutrients, nd Arseni Methyltion in West Bengl, Indi. Environ. Helth Perspet. 119, (11). 51. NNMB. Diet nd Nutritionl Sttus of Rurl Popultion. Report y Ntionl Institute of Nutrition. Indin Counil of Medil Reserh, Hyderd (). 5. Rosin, M. P., Zki, S., Wrd, A. J. & Anwr, W. A. Involvement of inflmmtory retions nd ell-profilertion in the development of ldder-ner in shistosomisis ptients. Mutt. Res. 35, 83 9 (199). 53. Bonssi, S. et l. An inresed mironuleus frequeny in peripherl lood lymphoytes predits the risk of ner in humns. Crinogenesis 8, (7). 5. Plöttner, S., Behm, C., Bolt, H. M. & Follmn, W. Effets of igrette smoke ondenste on primry urothelil ells in vitro. J. Toxiol. Env. Helth. Prt A. 75, (1). 55. Reli, D. et l. Mironulei in exfolited ells nd urine mutgeniity in smokers. Mutt. Res. 19, (1987). 56. Dunnett, C. W. Pirwise Multiple Comprisons in the Unequl Vrine Cse. J. Amer. Stt. Asso. 75, (198). 57. Knji, G. K. 1 Sttistil Tests. 3 rd Edition, SAGE Pulitions, London. 58. Lu, M. K. Dunnett-Tukey-Krmer Pirwise Multiple Comprison Test Adjusted for Unequl Vrines nd Unequl Smples Sizes. R Pkge DTK. Version 3.1, CRAN R repository. (13) Aessed 1 th June R Development Core Tem. R: A lnguge nd environment for sttistil omputing. R Foundtion for Sttistil Computing, Vienn, Austri. ISBN URL Version.15. (1) Aessed 1 th June De Mendiuru, F. Sttistil Proedures for Agriulturl Reserh. R Pkge griole. CRAN R repository. griole/griole.pdf Version 1.1-, (13) Aessed 1 th June 13. Aknowledgements The reserh ws supported y the British Counil funded UKIERI PRAMA projet (SA7-9) (DAP, AKG), CSIR (MB [NWP5], NB, PB), nd UK NERC Dorothy Hodgkins Postgrdute Awrd (DM). We thnk the following for ssistne with smple olletion (Dr. Jynt K. Ds, Dr. Apur K. Bndyopdhyy, Dr. Sntnu Bsu, nd Dr. Sptrshi Bnerjee), urine smple preprtion (Dr. Anirn Bnerjee nd Mr. Jynt Ds) nd rie smple preprtion (Seem Khttk, Cth Dvies, nd Willim Brdford). The views expressed here re not neessrily those of the funding odies or of ny individuls whose ssistne we knowledge here. Author ontriutions The study ws oneived jointly y A.K.G. nd D.A.P., who oordinted the smpling/ mironulei ssy nd hemil nlysis spets of the projet respetively. M.B., N.B., P.B., D.M. nd A.K.G. olleted the smples nd/or nlysed for MN under the supervision of A.K.G., M.B., N.B. nd P.B. ontriuted eqully to this work. P.L. undertook the instrumentl hemil nlyses. M.M. under the supervision of J.P. provided n independent sttistil nlysis of the dt susequent to its olletion. All uthors ontriuted to writing nd reviewing the mnusript. Additionl informtion Competing finnil interests: The uthors delre no ompeting finnil interests. How to ite this rtile: Bnerjee, M. et l. High rseni in rie is ssoited with elevted genotoxi effets in humns. Si. Rep. 3, 195; DOI:1.138/srep195 (13). This work is liensed under Cretive Commons Attriution 3. Unported liense. To view opy of this liense, visit SCIENTIFIC REPORTS 3 : 195 DOI: 1.138/srep195 8