Niek J. van Sittert a,), Peter J. Boogaard a, A.T. Natarajan b, Ad D. Tates b, Lars G. Ehrenberg c, Margareta A. Tornqvist d

Transcription

1 Mutation Researh Mutation Researh Frontiers Community address: Formation of DNA adduts and indution of mutageni effets in rats following 4 weeks inhalation exposure to ethylene oxide as a basis for aner risk assessment Niek J. van Sittert a,), Peter J. Boogaard a, A.T. Natarajan b, Ad D. Tates b, Lars G. Ehrenberg, Margareta A. Tornqvist d a Department of Moleular Toxiology, Shell International Chemials, Amsterdam, Netherlands b Department of Radiation Genetis and Chemial Mutagenesis, Leiden niõersity Medial Center, Leiden, Netherlands Department of Geneti and Cellular Toxiology, Stokholm niõersity, Stokholm, Sweden d Department of EnÕironmental Chemistry, Stokholm niõersity, Stokholm, Sweden Reeived 4 Otober 1999; aepted 12 Otober 1999 Abstrat Ethylene oxide EO. is mutageni in various in vitro and in vivo test systems and arinogeni in rodents. EO forms different adduts upon reation with DNA, N 7-2-hydroxyethyl. guanine N7-HEG. being the main addut. The major objetives of this study were:. a to determine the formation and persistene of N7-HEG adduts in liver DNA of adult male rats exposed to 0, 50, 100 and 200 ppm by inhalation 4 weeks, 5 daysrweek, 6 hrday. and b. to assess dose response relationships for Hprt gene mutations and various types of hromosomal hanges in spleni lymphoytes. N7-HEG adduts were measured 5, 21, 35 and 49 days after essation of exposure. By extrapolation, the mean onentrations of N7-HEG immediately after essation of exposure day 0. to 50, 100 and 200 ppm were alulated as 310, 558 and 1202 addutsr10 8 nuleotides, respetively, while the mean onentration in ontrol rats was 2.6 addutsr10 8 nuleotides. At 49 days, N7-HEG values had returned lose to bakground levels. The mean levels of N-2-hydroxyethyl- valine. adduts in haemoglobin were also determined and amounted 61.7, 114 and 247 nmolrg globin, respetively. Statistially signifiant linear relationships were found between mean N7-HEG levels day 0. and Hprt mutant frequenies at expression times 21r22 and 49r50 days and between mean N7-HEG day 0. and sister-hromatid exhanges SCEs. or high frequeny ells HFC. measured 5 days post-exposure. At day 21 post-exposure, SCEs and HFCs in-part persisted and were signifiantly orrelated with persistent N7-HEG adduts. No statistially signifiant dose effet relationships were observed for indution of mironulei, nor for hromosome breaks or transloations. In onlusion, this study indiates that following sub-hroni exposure, EO is only weakly mutageni in adult rats. sing the data of this study to predit aner risk in man resulting from low level EO exposures in onjuntion with other published data, i.e., those on. a genotoxi effets of EO in humans and rats,. b DNA binding of other arinogens,. natural bakground DNA binding and. d genotoxi poteny of low energy transfer LET. radiation, it is not expeted that ) Corresponding author. Shell Researh and Tehnology Centre, Amsterdam, P.O. Box 38000, 1030 BN Amsterdam, Netherlands. Tel.: q ; fax: q address: nio.j.vansittert@op.shell.om N.J. van Sittert r00r$ - see front matter q 2000 Elsevier Siene B.V. All rights reserved. PII: S

2 28 ( ) N.J. Õan Sittert et al.rmutation Researh long term oupational exposure to airborne onentrations of EO at or below 1 ppm EO produes an unaeptable inreased risk in man. q 2000 Elsevier Siene B.V. All rights reserved. Keywords: Ethylene oxide; Moleular dosimetry; N7-HEG; Hprt mutations; Chromosome aberrations; Natural bakground; Risk assessment; Radiation dose-equivalent 1. Introdution Ethylene oxide EO. is used primarily as a hemial intermediate in the prodution of ethylene glyol, non-ioni surfatants and other derivatives and is also used as a sterilising agent for medial devies. EO has shown to indue geneti damage, inluding gene mutations, hromosomal aberrations CA., mironulei MN. and sister-hromatid exhanges SCEs., in a wide variety of test systems suh as bateria and mammalian ells in vitro, somati and germ ells of rodents in vivo and lymphoytes from oupationally exposed workers w1,2 x. In long term inhalation studies EO indued brain tumours, mononulear ell leukaemia and peritoneal mesothew3 5 x, and lung adenomas and arino- lioma in rats mas, and ertain types of mammary and uterine tumours in the mouse wx 6. The indution of tumours in both speies was dose related but in brain, lung and uterus the frequeny was only statistially signifiantly inreased at the highest dose 100 ppm. of EO. The International Ageny for Researh on Caner IARC. lassified EO as ategory 1, Carinogeni in Humans. This lassifiation was heavily influened by published data on the ytogeneti damage in peripheral lymphoytes of human beings oupawx 7, but not based on epi- tionally exposed to EO demiologial studies with exess aner mortality or morbidity as endpoint. Reent epidemiologial data from 10 ohorts from five ountries, omprising 33,000 workers, indiate that EO does not lead to an inreased overall risk of aner, nor to an inreased risk of brain, stomah or panreati aners in partiular. The findings for leukemia and non-hodgkin s lymphoma are inonlusive. Exposure estimates from workers with longer follow up were on average between 5 and 15 ppm in hemial plants, but in sterilisation operations of medial equipment levels from ppm have been reported wx 8. The basis of the mutageniity and arinogeniity in rodents exposed to EO is onsidered to be its reativity with DNA, whih leads to the formation of different DNA adduts. The major addut is N 7-2- hydroxyethyl. guanine N7-HEG. and minor adduts 6 6 are O - 2-hydroxyethyl guanine O -HEG. and reation produts with N1, N3 and N 6 of adenine and with N 3 of ytosine, urail and thymine w9,10 x. N7-HEG is not onsidered to be promutageni in itself, as it does not ause base pair hanges and has no mispairing properties during DNA repliation. N7-HEG is suggested to be primarily involved in the indution of hromosomal aberrations w11 x. In addition, depurination of N7-HEG will lead to the formation of apurini sites whih may indue base pair w x 6 hanges 12. O -HEG is onsidered a misoding lesion probably due to mispairing with thymine during DNA repliation. In analogy with other monow13 x, it is expeted that funtional alkylating agents the ratio of the levels of N7-HEG and O 6 -HEG or other promutageni adduts is independent of EO exposure levels. Therefore, N7-HEG ould be used as a surrogate marker for other promutageni adduts, provided the relation between their onentrations has been established. Adduts are removed from the DNA in tissues by DNA repair proesses. For example, O 6 -HEG is effetively removed from the DNA by the repair protein O 6 -alkylguanine-dna alkylw14 x. Following inhalation exposure, EO is absorbed transferase effiiently into the blood and rapidly and widely distributed to all organs and tissues w15 x. Following both single and repeated exposure to EO, N7-HEG levels in DNA were similar in all tissues investigated, exept in testis where lower levels about 50% of other tissues. were measured w16,17 x. In these studies, alkylation of haemoglobin Hb. by EO was also measured. Reative sites in Hb were ysteine, N-terminal valine and N 1- and N 3-histidine w16 x. N-terminal valine HOEtVal. and N-histidine adduts have been used as biomarkers for monitoring of oupational exposure to EO w18 26 x. Levels of DNA and Hb adduts resulting from human exposure to EO an be onverted into umulative EO

3 ( ) N.J. Õan Sittert et al.rmutation Researh doses in blood and relevant target tissues target doses. w18 x. Genotoxiity data for EO have been used to predit geneti risk resulting from exposures to EO by using the parallelogram method w27 29x and aner risk by using the radiation dose equivaw30 x. In the latter, a relative poteny lene approah method is used whih ompares the genotoxi effets of EO with those of low LET radiation as referene standard w30 34 x. In these studies EO blood doses, derived from Hb adduts, were alulated and used as a parameter for EO doses in target organs. So far, little is known about the relationships between DNA adduts and the indution of mutation and aner by EO. By involvement of EO indued DNA modifiations N7-HEG. or EO target doses in dose response urves for simultaneously assessed parameters for geneti damage, a better aner risk assessment of low level exposure to EO in man an be ahieved. Therefore, the objetives of the present study were. 1 to measure the formation of N7-HEG in liver DNA of rats following 4 weeks exposures to EO ppm. via inhalation, 2. to evaluate the persistene of N7-HEG adduts,. 3 to establish the relationship between N7-HEG and HOEtVal adduts and to alulate EO target and blood doses derived from these adduts, and. 4 to onstrut dose response urves between N7-HEG adduts in liver DNA, EO blood doses or EO target doses and the indution of Hprt mutations, SCE, HFC, MN or hromosome breaks in splenoytes. Stable hromosome aberrations transloations., whih atually represent a greater geneti risk than the other ytogeneti parameters were also measured. N7-HEG was determined in liver beause higher yields of DNA will be obtained ompared to spleni or peripheral lymphoytes and, therefore, higher sensitivity of measurements of low levels of N7-HEG an be ahieved. From the published work disussed above, it is expeted that N7-HEG levels in liver, lymphow16,17,46 x. ytes and target organs are similar 2. Materials and methods 2.1. Animals Male Lewis rats n s 128. were shipped from Charles River Sulzfeld, Germany. to the inhalation faility in the Department of Mediine of the niversity Medial Centre in Trondheim, Norway. The animals were alimatised for three weeks prior to treatment and had random aess to food B& K niversal, Sweden. and tap water. At the beginning of exposure rats were young adults 8 9 weeks old. and weighed 256 g on average Inhalation exposure Rats were randomised into four groups of 32 animals and animals in eah group were equally distributed over four stainless steel ages, 8 ratsrage. Animals were exposed to EO via inhalation whole body. in three hambers with EO onentrations of 50, 100 and 200 ppm respetively. Controls were kept in another hamber. All exposures were performed for 6 h per day 9 AM to 3 PM., 5 days per week for 4 weeks w35,36 x. The onentration of EO was monitored and logged ontinuously by a gas sensorrtransmitter for EO. The hamber onentrations mean and SD. for the entire exposure period were 51"7, 100"15 and 200"25 ppm EO. Sixty-six hours after the termination of exposure, rats were shipped alive by plane and road to the Leiden laboratory where they arrived 96 h after exposure Colletion of speimens Whilst still alive, but deeply anaesthetised by inhalation of CO2 from evaporating dry ie, biologial speimens were olleted from the animals for analyses of the various assays at the following days after essation of exposure f. Table 1 for number of animals used per exposure group.. Day 5: Cytogeneti assays: spleni lymphoytes SPL. were isolated and stored frozen as desribed elsewhere for ytogeneti assays w35,36 x; HOEtVal adduts in Hb: blood samples were olleted from the vena ava, erythroytes were isolated and frozen as previously desribed w35 x, sent to Stokholm and stored at y208c until analysis; N7-HEG adduts in liver DNA: after bleeding the animals, livers were removed, frozen in liquid nitrogen and sent to Amsterdam and stored at y808c until analysis. Day 21 andror 22: Hprt mutations: Spleni lymphoytes SPL. were isolated and Hprt analysis was performed with fresh SPLs as desribed elsewhere

4 30 Table 1 Design of speimens olletion from male Lewis rats after essation of inhalation exposure to ppm Exposure Hb adduts Liver DNA adduts Hprt mutants SCE and HFC Mironulei Chromosome aberrations level Breaks Transloations ppm. day n day n day n day n day n day n day n N.J. Õan Sittert et al.rmutation Researh 447 ( 2000 ) n: number of rats; day: number of days after essation of exposure.

5 ( ) N.J. Õan Sittert et al.rmutation Researh w35 x; Cytogeneti assays and N7-HEG adduts in liver DNA as on day 5. Day 35 andror 36, 49, 50: Hprt mutations and N7-HEG adduts in liver DNA as on day 21r Hprt analysis The proedures for positive ontrols EN., isolation of lymphoytes from spleen SPL., priming and loning of SPL and alulation of mutant frequenies were desribed elsewhere w35 x Cytogeneti endpoints Cultures were set up from splenoytes for all biologial endpoints tested SCE, MN, CA.. Proedures for analysis of endpoints were desribed elsew36 x. where 2.6. Chemials for N7-HEG analyses Guanidinium hydrohloride, guanosine, 3-wN- morpholinoxpropane sulphoni aid MOPS., sodium hloride, sodium edetate dihydrate, tris-whydroxy- Tris. base, Tris HCl, methylxaminomethane trisodium itrate dihydrate, Triton-X 100 and Tween- 20 were of moleular biology grade and purhased from Sigma St. Louis, MO, SA.. Dihlorodimethylsilane, pentafluorobenzyl bromide, RNAse A and RNAse T1 were purhased from Sigma. Proteinase K was obtained from Boehringer Mannheim, Germany.. Glyidol was bought from Aldrih Milwau- kee, WI, SA.. The following reagents were bought from Merk Darmstadt, Germany. in the highest purity available: absolute ethanol, dihloromethane, ethyl aetate, ethylene oxide, fuming hydrohlori aid 37%., glaial aeti aid, n-hexane, potassium arbonate, potassium hydroxide, 2-propanol, siliagel mesh, 60 A. and toluene. Water used was deionised water further purified using a 50 Milli-Q mahine Millipore, Etten-Leur, The Netherlands Synthesis of standards N7-HEG was synthesised from guanosine and EO as follows: guanosine 2.0 g, 7.06 mmol. was suspended in 20 ml glaial aeti aid and ooled to 48C on ie. EO 1.4 ml, approximately 25 mmol. was added quikly and the suspension was subsequently shaken for 10 min at 48C. The vial was losed and stirred while heated to 378C overnight. The aeti aid was evaporated at 408C and the residue dissolved in 20 ml 1 M HCl and heated at 1008C for 1 h. The solution was ooled in ie and neutralised with 1 M NaOH to ph 5. The preipitate was filtered off, washed with ie-old water and aetone and subsequently dried in vauo. The dry residue was rerystallised from 350 ml hot water 958C., redissolved in hot water and deolourised with haroal, rerystallised again and dried in vauo over P2O5 for 48 h. A white rystalline substane was obtained at 72% yield. The V spetrum in H 2O on a Lambda 16 spetrophotometer, Perkin Elmer, Nieuwerkerk aan den IJssel, The Netherlands. showed lmax at and 283 nm. H-NMR in d6dmso using a Gemini 200 MHz mahine, Varian, Houten, The Netherlands.: d 3.70 q, N7`CH CH OH., t, N7`CH CH OH., 4.92 t, N7`CH CH OH , s, C `NH, 7.85 s, C `H, s, N -H. 2. The hemial ionisation mass spetrum showed a molewmqh q ular ion at mrz 196 x. N 7-2,3-dihydroxypropyl. guanine N7-HPG. was prepared from guanosine and glyidol as follows: guanosine 2.0 g, 7.06 mmol. was suspended in 30 ml glaial aeti aid 4.8 ml glyidol 58 mmol. was added. The vial was losed and the suspension was stirred for 17 h at room temperature. The aeti aid was evaporated at C, 50 ml of a mixture of aetone and diethyl ether 1:1. was added to the residue and the solvents were re-evaporated. The olourless, oily residue was dried on a vauum pump and subsequently dissolved in 20 ml 1 M HCl and heated at 1008C for 1 h. The solution was ooled in ie and neutralised with 1 M NaOH to ph 5. The preipitate was entrifuged 48C, 2500 = g., washed with water and aetone and subsequently dried in vauo. The dry residue was rerystallised from 100 ml hot water 958C., redissolved in hot water, filtered, and rerystallised again and dried in vauo over P2O5 for 48 h. A white rystalline substane was obtained at 76% yield. The V spetrum H 2 O. showed lmax at 246 and 287 nm. The identity of the produt was onfirmed by 1 H-NMR and mass spe- 1 trometry. H-NMR d DMSO, 200 MHz. 6 : d 3.82 m, N7`CH CHOHCH OH., 4.05 q, N7` 2 2

6 32 ( ) N.J. Õan Sittert et al.rmutation Researh CH CHOHCH OH., dd, N7`CH 2CH- OHCH OH., 4.75 t, N7`CH CHOHCH OH , d, N7`CH CHOHCH OH, 6.1 s, C `NH , s, C `H, 10.7 s, N `H.. The eletron impat w qø mass spetrum showed mrz 225 M x, base peak w qø x w qø mrz 151 guanine, mrz 207 M-H O x 2, mrz w qø x w qø 194 M-CH OH, mrz 165 N7-methylguanine x 2 and the hemial ionisation spetrum showed the moleular ion at mrz 226 wmqh q x DNA isolation DNA was isolated from liver using the following standard operating proedure: about 0.7 g of liver was mined with sissors and homogenised using a Braun homogeniser with strokes at 700 rpm in 38 ml aqueous buffer of 0.8 M guanidinium hydrohloride with RNAse A 54 rml. and T rml., 0.03 M Tris base, 0.03 M EDTA, 5% wrv. Tween-20 and 0.5% wrv. Triton-X100, adjusted with sodium hydroxide to ph 8.0. After addition of 2 ml proteinase K solution 164 rml., the homogenised suspension was inubated for 3 6 h at 378C in a shaking waterbath until the suspension beame lear. Following the inubation, the suspension was ooled down and stored overnight at y808c. Anion exhange olumns 500rG genomi tip; Qiagen, Hilden, Germany. were washed with 10 ml equilibration buffer 0.75 M sodium hloride in 15% aqueous ethanol with 0.15% wrv. Triton- X100, buffered with 50 mm MOPS, adjusted to ph 7.0 with sodium hydroxide. and half of the suspension 20 ml maximum. was eluted through the olumn by gravity, the other half was eluted through a seond olumn. The olumns were subsequently washed twie with 15 ml wash buffer 1.0 M sodium hloride in 15% aqueous ethanol, buffered with 50 mm MOPS, adjusted to ph 7.0 with sodium hydroxide. at room temperature. Finally, the DNA was eluted from the olumns by washing with 15 ml elution buffer 1.25 M sodium hloride in 15% aqueous ethanol, buffered with 50 mm Tris adjusted to ph 8.5. at 378C. The DNA solutions from both olumns were pooled and the DNA was then preipitated by addition of half a volume of isopropanol and olleted by entrifugation at 11,000 = g and 48C for 15 min. The pellet was washed with 4.0 ml of ie-old 70% wrv. ethanol and entrifuged one more at 10,000=g and 48C for 15 min. The pellet was then lyophilised. After dissolution of the dry pellet in a small volume of purified water, the DNA onentration was determined by V-measurements at ls260 and 280 nm, assuming that A260 s20.4 for a DNA solution of 1 mgrml at a path length of 10 mm Measurement of N7-HEG N7-HEG was released form the DNA bakbone by neutral thermal hydrolysis depurination.. The DNA solution was heated to C for 1 h in a Thermomixer 5437 Eppendorf, Hamburg, Germany.. After ooling to room temperature, the samples were entrifuged at 3000=g for 2 min and the released adduts were isolated by miro-ultrafiltration through a filter with a moleular weight ut-off of 3000 Dalton Centrion-3, Amion, Etten-Leur, The Netherlands. at 6500 = g and 48C for 2 h. The filtrates were lyophilised in silanised Reati-vials w. For analysis the residue was dissolved in 100 ml 0.1 M HCl and 50 ml of a 2.0 mm solution of N7-HPG in 0.1 M HCl was added as internal standard. The samples were evaporated to dryness under a gentle stream of nitrogen in a heating blok thermostated at 358C. The dry residue was pentafluorobenzylated aording to Saha et al. w37,38 x. Briefly, the vials were plaed on ie and the residues dissolved in 50 ml 6 M HCl degassed with nitrogen for 5 min. and the guanidinium moiety oxidised with 20 ml tertbutyl nitrite while stirring 4 h at 08C. The solvent was evaporated under nitrogen, 150 ml ethyl aetate and 50 ml water were added, the stirrer bar was removed and washed with 50 ml water whih was added to the sample. The Reati-vials w were entrifuged at 1800=g for 2 min and the aqueous layer olleted and evaporated to dryness on a heating blok thermostated at 358C. To the dry residue 5 mg of dry, powdered potassium arbonate and 100 ml of a solution of 100 ml pentafluorobenzyl bromide in 1 ml aetonitrile were added. After stirring the mixture for 20 h at room temperature, the samples were entrifuged at 1800=g for 2 min and evaporated to dryness under nitrogen. Then, 50 ml of a solution of 2.5 mgrml tetrabutylammonium hydrogensulphate in 1 M KOH, 150 ml dihloromethane and 5 ml pentafluorobenzyl bromide were added and the mixture was stirred at room temperature for another 20 h. At the end of that period, the samples were

7 ( ) N.J. Õan Sittert et al.rmutation Researh entrifuged at 1800 = g for 2 min and the dihloromethane was evaporated slowly under nitrogen. The residue was extrated three times with 200 ml ethyl aetate, the ombined ethyl aetate frations were evaporated under nitrogen and the residue was dissolved in 25 ml ethyl aetate and 25 ml n-hexane was added. The samples were applied on siliagel olumns 500 mg, prepared in silanised glass pasteur pipettes plugged with glass wool prior to silanisation. whih were freshly washed with 1 ml ethyl aetate and 1.5 ml n-hexane prior to appliation. The olumns were eluted with 4.5 ml n-hexane and subsequently with 6.0 ml 10% vrv. ethyl aetate in n-hexane. The derivatised produts were finally eluted from the olumns with 2 ml ethyl aetate into lean, silanised Reati-vials w. The eluate was evaporated to dryness under nitrogen and the dry residue dissolved in 50 ml toluene for analysis by GC-MS. For eah series of analysis, alibration standards were prepared by addition of known amounts of N7-HEG to 1 mg of untreated alf thymus DNA, whih was subsequently derivatised as desribed above. The amount of added N7-HEG ranged from 0.11 to 400 pmol. A plot of the logarithm of the ratio of the peak area of N7-HEG and the peak area of the internal standard N7-HPG. as a funtion of the logarithm of the ratio of the onentrations of N7- HEG and N7-HPG was used as a alibration urve. The alibration urve was linear over the entire onentration range. The limit of quantifiation was determined at 0.2 addutsr10 8 nuleotides for a 1 mg DNA sample. The reproduibility between assays day-to-day variation. was determined by inlusion of the same referene samples ontaining 1 mg DNA with 4 pmol or 0.1 nmol N7-HEG and eah 0.1 nmol N7-HPG. in eah series of analyses. The oeffiients of variation n s 10. were 46% 4 pmol level. and 19% 0.1 nmol level.. Analyses were performed using a 5890 gas hromatograph Hewlett Pakard, Amstelveen, The Netherlands. on a 0.25 mm internal diameter DB-5- MS apillary olumn J and W Sientifi, Folsom, CA, SA. with a film thikness of 0.10 mm. The head pressure was kept at 65 kpa. Samples 1 ml, splitless. were introdued with a programmable temperature vaporiser Gerstel, Mulheim an der Ruhr, Germany.. The ompounds were eluted from the olumn using the following oven programme: initially 908C for 1 min, then an inrease to 2508C at 208rmin followed by an inrease to 3508C at 108rmin, the temperature of 3508C was kept for 5 min. For detetion a 5989 mass spetrometer Hewlett Pakard. was used either in the eletron impat mode EI. or in the negative hemial ionisation mode NCI.. For EI the interfae and the soure temperature were kept at 2608C, sans were made of the range amu at a rate of 0.9 sansrs. For NCI EI the interfae and the soure temperature were kept at 3008C, sans were made of the range amu at a rate of 0.9 sansrs. For quantitation, the NCI mode was used with seletive ion monitoring of the ions 555 quantitation. and 535 qualifier. for the pentafluorobenzyl derivatives of N7-HEG and of the ions 765 quantitation. and 745 qualifier. for the pentafluorobenzyl derivatives of N7-HPG internal standard Analysis of HOEtVal in Hb HOEtVal in Hb was determined as desribed by Tornqvist w39 x. Briefly, globin was preipitated with ethyl aetate from an isopropanolrhcl solution and the modified N-terminal valine in Hb was speifially leaved and isolated as pentafluorophenyl thiohydantoin after derivatisation of globin samples with pentafluorophenyl isothioyanate. Globin alkylated with deuterium substituted EO was added before derivatisation as internal standard for the quantifiation of addut levels. Calibration samples were prepared from globin with a known addut level of HOEtVal. Addut levels in samples were quantified by mass spetrometri analyses using a Finnigan GCQ or FSQ 700 mass spetrometer. The instruments were operated in the negative ion hemial ionisation NICI. mode with methane as reagent gas and argon as ollision gas Calulations and statistial analysis The dose, D, in a tissue is defined as the integral of the onentration of a reative hemial or metabolite over time dimension: mol = L = h or Mh. w18,31 x. In the present study, the EO target dose D. target was derived from N7-HEG in liver DNA and the EO blood dose D. blood from HOEtVal in Hb. The amount of N7-HEG mol = g DNA. formed in tissue DNA is the produt of D Mh. target

8 34 ( ) N.J. Õan Sittert et al.rmutation Researh and the rate onstant k L=g =h. gua for the reation of EO with N7 of guanine in DNA. The amount of HOEtVal mol g globin.. formed in Hb is the produt of D blood and the rate onstant k val L = g = h. for the reation of EO with N- terminal valine in Hb. D is alulated from the formula: DsA um =k, in whih Aum is the umulative amount of addut formed during the 26 days exposure period. For an addut to DNA with first order kinetis of elimination, Aum s A ss = k el= t, in whih Ass is the steady state addut level and kel the rate onstant for loss of DNA adduts due to DNA repair andror hemial depurination w18 x. Following 26 days exposure A sa =0.178 d. =26 d. um ss s4.6=a ss. The target dose, D target, is then alulated from the formula: D Mh. sn7-hegmol= g DNA.. target = 4.6 = k L= g = h. gua, in whih N7- HEG is the amount of addut measured a short time after the last exposure on day 26, at whih time a steady state will be approahed w40 x. For an addut to haemoglobin with zero order kinetis of elimination, A sa=1r 1ytr2t. um er, in whih t is the life span of erythroytes er s 60 days in rats and 126 days in humans. w18,41 x. Following 26 days exposure Aum s1.3=a 26 days. The blood dose, D blood, is then alulated from the for- mula: D Mh. shoetval mol= g globin.. blood =1.3= k L=g =h. val, in whih HOEt- Val is the amount of addut measured a short time after the last exposure on day 26. Differenes between exposed and ontrol groups with respet to dosimetry and geneti parameters were assessed using one way analysis of variane. Signifiane of differenes was tested at the 5% level. Pearson s orrelation oeffiients were omputed to assess the linearity of relationships. Signifiane of linearity was tested at the 5% level. Regression analysis was used to quantify the dose response relationships between:. 1 EO exposure and N7-HEG or HOEtVal,. 2 N7-HEG and HOEtVal,. 3 EO exposure and Hprt, SCE or HFC,. 4 N7-HEG and Hprt, SCE or HFC and. 5 D blood or Dtarget and Hprt, SCE or HFC. The interepts were set at zero if they did not signifiantly differ from zero. For Hprt mutant frequenies, the doubling dose was assessed, i.e., the EO exposure for whih a doubling of the Hprt bakground value in ontrol rats was found. The doubling dose was alulated from the regression lines between EO exposure or N7-HEG day 0. and Hprt mutant frequeny after expression times of 21 and 22 days, using the bakground mutant frequeny of the onurrent ontrol group f. Table 7.. Its 95% onfidene limits were dedued from the 95% onfidene limits of the regression lines. 3. Results 3.1. N7-HEG in liõer tissue The mean levels of N7-HEG in liver DNA from rats following exposure to ppm EO by inhalation for 4 weeks and sarified 5, 21, 35 and 49 days 200 ppm only. after essation of EO exposure are shown in Table 2. Statistially signifiant differenes were observed in N7-HEG onentrations between all exposure groups. N7-HEG disappeared slowly from liver DNA. At day 21 after essation of expo- Table 2 Amounts of N7-HEG in liver DNA from Lewis rats, determined 5, 21, 35 and 49 days after essation of EO exposure ppm, 4 weeks, 5 daysrweek, 6 hrday. 8 Exposure ppm N7-HEG in liver DNA addutsr10 nuleotides. 5 days 21 days 35 days 49 days n mean SD n mean SD n mean SD n mean SD n: number of rats; days: number of days after essation of exposure; SD: standard deviation.

9 ( ) N.J. Õan Sittert et al.rmutation Researh Table 3 Mean N7-HEG levels in liver DNA from Lewis rats, determined 5 days after essation of exposure to EO ppm for 4 weeks, 5 daysrweek, 6 hrday. and extrapolated bak to day 0 after exposure Exposure ppm. N7-HEG in liver DNA EO target dose mmh. 8 addutsr10 nuleotides. y5 y5 gua gua gua 5 days Extrapolated bak to day 0 k s5.17=10 k s10=10 k a b T s7 days T s3.9 days average. 1r2 1r Target doses were alulated from addut levels at day 0 and T 1r2 s3.9 days. 1 addutr10 nuleotidess0.324 nmol= g DNA. Values in brakets denote the standard deviation; day. s : number of days following essation of exposure. a Present study. b Walker et al. w17 x. Combined ontrol levels 5, 21 and 35 days after exposure. Signifiantly different from ontrol group P sure, N7-HEG levels were about 20% of levels measured at day 5 for all exposure levels tested. At 49 days after essation of exposure, the onentrations of N7-HEG in rats exposed to 200 ppm EO had returned lose to bakground levels as established in ontrol rats. The amounts of N7-HEG in liver DNA immediately after ompletion of exposure day 0. an be alulated from addut measurements at day 5 after exposure and the in vivo half-life for N7-HEG, assuming first order kinetis of loss of these adduts from DNA. The in vivo half-life for N7-HEG in liver DNA, determined from addut measurements 5 and 21 days after the end of exposure was about 7 days at all EO exposure levels tested. A half-life of 3.9 days was reported for N7-HEG in liver DNA of F344 rats, exposed to 300 ppm EO for 4 weeks w17 x. This half-life was alulated from N7-HEG analyses of liver DNA in rats killed 0, 1, 3 and 7 days after essation of exposure and is probably more aurate than the alulation from N7-HEG in rats killed 5 and 21 days post-exposure in our study. In Table 3 the alulated amounts of N7-HEG in liver DNA at day 0 and the orresponding target doses D target. are shown. N7-HEG levels alulated from a half-life of 3.9 days are used in the alulation of target doses and in the dose response relationships between N7- HEG at day 0 and geneti parameters investigated in this study. Target doses were alulated using Table 4 Signifiant relationships between exposure and dosimetry parameters X Y No. of Slope SE. Multiple a P Regression data points R line Exposure b N7-HEG Fig. 1 Exposure b HOEtVal Fig. 1 N7-HEG HOEtVal d N7-HEG d HOEtVal Interepts were set at zero if they did not signifiantly differ from zero. nits: Exposure in ppm; N7-HEG in addutsr10 8 nuleotides; HOEtVal in nmolrg globin. a P-value of the null hypothesis that the slopes0. b Individual measurements determined 5 days after essation of exposure, extrapolated bak to day 0 post-exposure. Mean values per exposure group 0, 50, 100 and 200 ppm., determined 5 days post-exposure. d Mean values per exposure group determined 5 days after essation of exposure, extrapolated bak to day 0 post-exposure.

10 36 ( ) N.J. Õan Sittert et al.rmutation Researh were found in HOEtVal levels between all exposure groups. From these HOEtVal levels, the extent of HOEtVal in globin at day 0 and the orresponding blood doses D. blood were alulated, assuming zero order kinetis of HOEtVal elimination Table 5.. Blood doses were alulated using published reation rate onstants k. w41,44 x val. There was a highly signifiant linear relationship p between EO exposure ppm range. and HOEtVal levels at either day 0 or day 5 post-exposure Table 4.. The regression lines for HOEtVal onentrations at day 0 are shown in Fig. 1. Fig. 1. Formation of DNA adduts N7-HEG, v. and Hb adduts HOEtVal, `. of EO in male Lewis rats as funtion of exposure to EO by inhalation during 4 weeks, 5 daysrweek, 6 hrday. N7-HEG and HOEtVal values were extrapolated bak to day 0. Solid lines represent the average regression lines and the dashed lines the tolerane limits of the 95% onfidene intervals for the average values. The interepts were set at zero sine they did not signifiantly differ from zero ps0.941 and 0.882, for N7-HEG and HOEtVal, respetively.. published reation rate onstants k. w42,43 x gua. A highly signifiant linear relationship was found between EO exposure ppm range. and N7-HEG levels at days 0, 5 and 21 post-exposure Table 4.. The regression lines for N7-HEG onentrations at day 0 are shown in Fig HOEtVal in Hb The mean onentrations of HOEtVal in Hb from rats following exposure to ppm EO and sarified 5 days after essation of EO exposure are shown in Table 5. Statistially signifiant differenes 3.3. Relationship between DNA and Hb adduts HOEtVal onentrations were determined in globin from other rats than those used for N7-HEG analysis. Therefore the relationship between HOEt- Val addut formation in Hb and N7-HEG addut formation in liver DNA following exposure to EO ould only be investigated using their mean values per exposure group 0, 50, 100, 200 ppm.. Highly signifiant linear relationships were found between HOEtVal and N7-HEG in the ppm range, either at day 0 or day 5 post-exposure Table 4.. The EO doses in liver and blood, derived from N7-HEG and HOEtVal, respetively, were also signifiantly orrelated. The ratio D target: D blood was on average f. Tables 3 and Hprt mutations Results of indution of Hprt mutations in SPL from rats following EO exposure and neropsied 21 Table 5 Mean HOEtVal levels in globin from Lewis rats, determined 5 days after essation of EO exposure ppm for 4 weeks, 5 daysrweek, 6hrday. and extrapolated bak to day 0 after exposure Exposure ppm HOEtVal in globin nmolrg globin EO blood dose mmh y5 y5 val val val n 5 days extrapolated bak to k s4.19=10 k s5.8=10 k day 0 w40x w44x average Blood dose alulated from addut levels at day 0. Values in braket denote the standard deviation. Signifiantly different from the ontrol group P

11 Table 6 Data on exposure and Hprt mutant frequeny and ytogeneti effets SCEs, HFC, Mironulei, Chromosomal aberrations breaks and transloations Exposure Hprtr10 SCEr HFC MNr10 CA CA trans- ppm. ells ell %. ells breaks. r loations. r 2 10 ells 2 10 ells Days after exposure: 21, n mean n mean n mean n mean n mean n mean n mean n mean SD. SD. SD. SD. SD. SD. SD. SD a b b b b n: number of rats; days: number of days after essation of exposure; SD: standard deviation. a Combined ontrol values after 21r22, 35r36 and 49r50 days used for statistial analyses between the groups.. b Combined ontrol values 5 and 21 days post-exposure used for statistial analyses between the groups.. Signifiantly different from ontrol group p N.J. Õan Sittert et al.rmutation Researh 447 ( 2000 )

12 38 ( ) N.J. Õan Sittert et al.rmutation Researh Table 7 Signifiant relationships between exposure or dosimetry parameters and geneti parameters X Y No. of Interept Slope Multiple b P Regression data SE. SE. R line points a Exposure Hprt 21, 22 days d D Hprt 21, 22 days blood d D Hprt 21, 22 days target d N7-HEG Hprt 21, 22 days Fig. 2 Exposure SCE 5 days d D SCE 5 days blood d D SCE 5 days target d N7-HEG SCE 5 days Fig. 3 e N7-HEG SCE 5 days Fig. 4 f N7-HEG SCE 21 days Fig. 4 Exposure HFC 5 days d N7-HEG HFC 5 days Fig. 3 e N7-HEG HFC 5 days Fig. 5 f N7-HEG HFC 21 days Fig. 5 nits of parameters: see Table 3,5,6. a Number of individual measurements for geneti parameters. b P-value of the null hypothesis that the slopes0. Days post exposure that geneti parameters were measured. d Mean values per exposure group determined 5 days after essation of exposure, extrapolated bak to day 0. e Individual measurements determined 5 days after essation of exposure. f Individual measurements determined 21 days after essation of exposure. or 22 days after essation of EO exposure are shown in Table 6. At days 21r22, statistially signifiant differenes were observed in mutant frequeny between the 200 ppm group and the ontrol group P Linear regression analyses showed a signifiant exposure, blood or target dose related inrease of Hprt mutant frequeny Table 7.. The relationship between N7-HEG levels in liver DNA at day 0 mean values per exposure group. and Hprt mutant frequeny 21r22 days. was also statistially signifiant Table 7.. The regression line is shown in Fig. 2. In the present study no analysis was performed on Hprt indution in rats neropsied 35r36 or 49r50 days after essation of exposure, beause previous studies showed the strongest mutageni efw35 x. fet at 21r22 days SCE frequeny at all exposure levels tested ompared to the ontrol group P Regression analysis showed a signifiant exposure, blood or target dose related effet on the frequeny of SCE 3.5. SCEs and HFC Results of the indution of SCE in SPL from rats sarified 5 and 21 days after essation of exposure are shown in Table 6. At day 5 post-exposure, statistially signifiant inreases were observed in Fig. 2. Hprt mutant frequeny at day 21r22 as funtion of level of DNA adduts of EO, expressed as the average hepati onentration N7-HEG extrapolated bak to day 0, in male Lewis rats exposed to EO by inhalation during 4 weeks, 5 daysrweek, 6 hrday.

13 ( ) N.J. Õan Sittert et al.rmutation Researh Fig. 3. SCE - Ø- and HFC -(- frequenies at day 5 as funtion of the average levels of DNA adduts of EO, expressed as hepati onentration N7-HEG on day 0, in male Lewis rats exposed to EO by inhalation during 4 weeks, 5 daysrweek, 6 hrday. Table 7.. A signifiant linear relationship was also found between N7-HEG levels in liver DNA at day 0 mean values per exposure group. and indution of SCEs Table 7 and Fig. 3.. In six rats sarified 5 days after exposure, N7-HEG was determined in liver DNA obtained from the same rats that were used for SCE analysis. Statistial analysis of these data also showed a signifiant linear orrelation between N7-HEG levels at day 5 post-exposure and indution of SCEs Table 7 and Fig. 4. The results of high frequeny SCEs HFC. are also shown in Table 6. The HFC threshold was defined as the 95% upper onfidene limit of SCE distribution for the ontrol data. At day 5 post-exposure, HFCs were signifiantly inreased at the 50 and 200 ppm groups ompared to the ontrol group p Signifiant linear relationships were found between exposure or N7-HEG levels at day 0 mean values per exposure group. and indution of HFC Table 7 and Fig. 3.. N7-HEG adduts and HFCs, measured in the same six animals 5 days after the end of exposure, were also signifiantly orrelated Table 7 and Fig. 5.. Between days 5 and 21 after essation of exposure, the SCE frequeny delined but was still signifiantly higher P at day 21 post-exposure ompared to the ontrol group for EO exposures of 100 and 200 ppm. The frequeny of HFCs also delined between days 5 and 21 post-exposure, but remained signifiantly higher at day 21 post-exposure at the 100 and 200 ppm exposure level P N7-HEG adduts measured 21 days following essation of exposure were signifiantly orrelated with both SCEs and HFCs measured 21 days post-exposure Table 7 and Figs. 4 and Mironulei and hromosome aberrations The data for mironulei MN and hromosome aberrations in SPL are shown in Table 6. Statistially Fig. 4. SCE frequeny at day 5 -Ø- and day 21 -(- as funtion of the orresponding individual levels of DNA adduts of EO, expressed as hepati onentration N7-HEG on day 5 and 21, in male Lewis rats exposed to EO by inhalation during 4 weeks, 5 daysrweek, 6 hrday. Fig. 5. HFC frequeny at day 5 -Ø- and day 21 -(- as funtion of the orresponding individual levels of DNA adduts of EO, expressed as hepati onentration N7-HEG on day 5 and 21, in male Lewis rats exposed to EO by inhalation during 4 weeks, 5 daysrweek, 6 hrday.

14 40 ( ) N.J. Õan Sittert et al.rmutation Researh signifiant differenes were found neither for the indution of MN and hromosomal breaks nor for transmissible hromosome aberrations reiproal transloations. between the EO exposed groups and the ontrol group. No signifiant relationships P) were found between exposure, blood dose or N7-HEG adduts and frequeny of MN, hromosome breaks or reiproal transloations. 4. Disussion The major aim of the present study was to investigate the dose response relationship of the formation and persistene of N7-HEG adduts in liver DNA with the indution of geneti damage in spleni lymphoytes from Lewis rats following 4 weeks inhalation exposure to EO. This will allow more insight into the qualitative andror quantitative role of DNA adduts in EO-indued mutagenesis and arinogenesis in rats and form the basis for a aner risk assessment Dosimetry In the present multi-site study, for logisti reasons, the first olletion of speimens was only possible 5 days after essation of exposure. Published data on the kinetis of DNA addut formation following exposure to monofuntional alkylating agents show that the highest onentrations are reahed a few hours following essation of exposure w13,17 x. Therefore, N7-HEG values measured in liver tissues olleted 5 days after essation of EO exposure were extrapolated bak to day 0, using a biologial half-life for N7-HEG loss of 3.9 days w17 x. In the present study, a half-life of about 7 days was alulated from the time dependent deline in the N7-HEG levels measured 5, 21 and 35 days post-exposure. These data indiate that N7-HEG removal from liver DNA might our in a bi-phasi way, i.e., a rapid first phase 0 5 days post-exposure. with a half-life of 3.9 days, followed by a slower seond phase of about 7 days. The dose response for N7-HEG in liver DNA extrapolated bak to day 0. was linear between 0 and 200 ppm EO Fig. 1., whih is in aordane with reent results from Wu et al., who found linearity for N7-HEG in liver DNA from F344 rats exposed to EO onentrations between 0 and 100 ppm w46 x. These results suggest that detoxifiation and DNA repair were not saturated at EO exposures up to 200 ppm. The dose response for HOEtVal ex- trapolated bak to day 0. was also linear between 0 and 200 ppm EO, whih onfirms that detoxifiation was not saturated at 200 ppm EO. The levels of N7-HEG and HOEtVal in our study are similar to those found in F344 rats exposed to 100 ppm EO w17,45,46 x. The effet of low level EO exposure on N7-HEG levels an be alulated using the regression line in Table 4. For example, in Lewis rats inhalation exposure of 1 ppm EO for 4 weeks leads to a alulated steady state level of N7-HEG of 5.9 addutsr10 8 nuleotides upper limit of the 95% onfidene inter- 8 val: 6.4 addutsr10 nuleotides. whih is about twie the level of endogenously produed N7-HEG 8 of 2.6 addutsr10 nuleotides Table 3.. These bakground N7-HEG levels are omparable with levw46,47 x. With els in ontrol rats reported by others regard to HOEtVal, a level of about 1.22 nmolrg globin upper limit of the 95% onfidene interval: 1.33 nmolrg globin. is produed following 4 weeks exposure to 1 ppm EO, whih is 25- to 30-fold higher than the onentration of endogenously produed HOEtVal of nmolrg globin Table 5.. In the present study EO blood doses D blood. derived from HOEtVal were on average 2.5-fold higher ompared to EO doses in liver tissue D target. derived from N7-HEG over the range of ppm EO. This ratio ompares favourably with the ratio of 1.5, found following single exposures to EO in the range of 0 33 ppm w16 x. EO doses in other tissues have shown to be similar to that in liver w16,17,46 x. From these data, it an be onluded that following single or repeated EO exposure, the EO blood doses are of the same order of magnitude ompared to EO doses in tissues. Therefore, this study onfirms that HOEtVal adduts an be used to monitor blood doses and tissue doses for EO risk assessment over a broad range of EO exposures. The time of speimen olletion after essation of exposure has to be taken into aount, as 5 days after the end of exposure the ratio HOEtVal: N7-HEG was 2.2-fold higher ompared to day 0 Table 8.. The influene of EO exposure level and of duration of exposure on HOEt-

15 ( ) N.J. Õan Sittert et al.rmutation Researh Table 8 Ratio HOEtVal in Hb and N7-HEG in liver DNA from rats from three studies Exposure Duration HOEtVal r N7-HEG Referene onentration pmolrg Hbradduts ppm. 8 per 10 nuleotides weeks, 5 daysrweek, 6 hrday 450 present study b 204 present study weeks, 5 daysrweek, 6 hrday 124 w17,45x weeks, 5 daysrweek, 6 hrday 60 w48x day, 6 h 40 w16x a HOEtVal and N7-HEG determined 5 days after essation of exposure. b HOEtVal and N7-HEG determined 5 days after essation of exposure, extrapolated bak to day 0 post- exposure half-life: 3.9 days. HOEtVal and N7-HEG determined about 2 h after essation of exposure. a Val: N7-HEG ratio is also shown in Table 8. An advantage of the use of HOEtVal as a monitor for EO exposure, ompared to N7-HEG, is its greater sensitivity for monitoring populations exposed to low levels of EO, beause its analytial limit of detetion is 1 pmolrg globin while for N7-HEG it is 0.2 adduts in 10 8 nuleotides for a 1 mg DNA sample. From the regression line day 0 post-exposure. in Table 4, it an be alulated that an 8 inrease of 0.01 N7-HEG addutr10 nuleotides 1 addut per ell. orresponds with an inrease of about 2 pmol HOEtValrg globin Hprt mutations In our study, a weak statistially signifiant dosedependent inrease in Hprt mutant frequeny was found in adult Lewis rats for effets measured after expression times of 21r22 days. Only one of the 24 Hprt analyses from the exposed groups ppm., with a mutant frequeny of 11.4, fell outside y6 the range of values = 10. of a historial ontrol group. However, 4 Hprt results were outside the range of the onurrent ontrol group. The data indiate that no detetable effet ould be observed at or below the 100 ppm exposure level. The weak mutageni effet of EO in adult Lewis rats was also demonstrated following experiments where rats were dosed with EO autely through single intra-peritoneal injetions or hronially in drinking water w35 x. The EO exposure that aused a doubling of the mutant frequeny of the onurrent ontrol group in the present study was estimated as 170 ppm 95% onfidene interval: ppm. or 20,400 ppmh ,000 ppmh. whih is 3.3-fold higher ompared to the doubling dose of 6000 ppmh alulated for adult male B6C3F1 lai transgeni Big Bluee. mie in a omparable 4 weeks inhalation study w28 x. Hprt mutant frequenies in T-lymphoytes from mouse spleen ells inreased by a fator 1.7, 3.1 and 6.4 over the ontrol frequeny in exposure groups 50, 100 and 200 ppm respetively, whih is higher ompared to the inrease found in Lewis rats in our study w49 x. In rats, the doubling dose of N7-HEG in liver DNA was about 1000 adduts per 10 8 nuleotides per 10 nuleotides. O -HEG adduts are promutageni and onsidered more relevant than N7-HEG for the indution of Hprt mutations. In F344 rats, following 4 weeks EO exposure, O 6 -HEG levels were tentatively deteted and estimated fold lower ompared to N7-HEG w17 x. The alulated O 6 -HEG level assoiated with a doubling of the spontaneous Hprt mutant frequeny was, therefore, about 4 5 adduts per 10 8 nuleotides. The role of O 6 -alkylguanine lesions in the indution of Hprt gene mutations has been studied with the model monofuntional ethylating agent ethyl methanesulfonate EMS. whih, like EO, reats mainly with guanine-n7 and to a minor extent with guanine-o 6. Indiret evidene for the important role of O 6 -ethylguanine in EMS-indued mutagenesis was obtained from the mutational spetrum of EMS-indued Hprt mutations in rat T-lymphoytes, sine GC to AT transitions were found at high frequeny w50 x. Similar lasses of mutations in T-ell mutants from mie following treatment with high doses of