Document ZnoORnxBdYxb20qBdqjejVaaY

i tfi r^iWriii fcafrtfdrfrrfl Ifrfl-fffilrt Canctr i.tntrt, 25(1981) 13-18 Elsevier Scientific Publishers Iretand Ltd C!jSr,'VVfr-()(r. o'.'OPY ^INDUCTION OFSlNCLE-STRANEUiUEAKS IN DNA OF MICE AFTER,'. -INHALATION OF VINYL CHLORIDE =^5.__ "' S.A .LS__O_L_V__E_I_C_W__A__L_L_E_S--a-m---l--B--O--,-H--O---L-M---B--E--R--G---- '') C. Ft-.. . Ortuyofwia(T<w)io^y,'Retfiareft Dep^me/it, Ma(j6n|( Board vf~ Occujiftionet'Safriy-wtl Health, S-f 71 84 Solti a -- 1 J_ (Received 2G July 1984) (stT" (R evised version received 30 August 1981) (Accepted 13 September 1984) z SL'.MMAR V Female mice u ere exposed to 500 ppm vinyl chloride fVC) for 6 h day 5 days/week for 1--8 weeks. Groups of mice were killed at different times during this period. DNA damage, expressed as singtestrand breaks (SSB), was studied in liver, kidneys, lungs, spleen and brain. The level of SSB increased in liver, kidneys, spleen and lungs with time of exposure and reacned a plateau tor kidneys and lungs after 80 and 120 h of exposure, In spleen there was only a slight increase in the SSB. and In brain no detect able increase was found. INTRODUCTION VC is mutagenic to several organisms and carcinogenic to humans, mice, rats and hamsters [ 1J, In mice it produces tumors in several organs, e.g. the liver, lungs, kidneys and mammary glands (1--3). ` VC is rapidly metabolized by a monooxygenase enzyme to chlorocthyjene oxide 14). Chloroethylene oxide can spontaneously transform to chloroacelaldehyde. Both these compounds arc electrophilic agents and react with proteins and nucleic acids in vivo [5]. in this study the induction of SSB have been studied in the DNA of female mice after inhalation of 500 ppm VC. The results show that VC induces SSB in DNA in the liver, kidneys, spleen and lungs, M VrEJllAI.8 AND METHODS Chctnicats VC (> 99.991) from AG A, Sweden, was used, llydroxylapatite (Bio-Gel 111 P and DNA-grade Bio-Gel 11TP Bio Rad Laboratories, Richmond, CA, 030 I 3835(8-1 /S03 UO E 1964 Elsevier Scientific Publishers I re] an cll.td. Published and Primed in Ireland If 14 tabu: i EXPOSURE SCHEME Exposure time (h> No of animals Mice killed 2 h after ex posure Mice killed 3 H h after ex posu rc 39 CO ni 234 36 11 1 231 4 4 4 4 4 4 0 Control animals, exposed 10 air only, were killed a Tier 36 h [n = 3} and 231 h (n k l). U.S.A.) and 4,6-diamidino-2-phenylindo!e 2HCJ (Serva-Feinbioehermca, Heidelberg, F.R.G.) were purchased. Artmiaj'.s and exposure Female mice (strain NMR1, 5 weeks old) were used, The mice were exposed for 6 h/day 5 days/week in 10-C desiccators with 15 animals in each. The temperature in the desiccators was 244C, the relative humidity 60--70*7 and the airflow 4 t/min The VC gas was diluted with air to a concentration of 500 ppm. The VC concentration was checked continuously with a Miran-1 A infrared spectrophotometer with a recorder Control animals were exposed to air only. Animals were killed by cervical dislocation 2 or 18 h after the exposure period, and immediately dissected for SSB determination. Table 1 shows the exposure scheme. Determination of ZJ.V.4 -strand breaks Cell nuclei were prepared from liver,, kidney, lung, spleen and brain according to the method described by Walles and Orsen [6]. SSB were determined by the DNA-unwindmg technique of Ahnstrom and Erixon (7] as modified by Walles and Erixon [8). The fraction (Fqs) of doublestranded DNA (DS) was calculated [9] as the ratio of the amount of DS and the sum of DS and single-stranded DNA (SS): DS Fds DS 4 SS The negative logarithm of Fps is a linear function of the number of SSB. RESULTS The results are presented in Fig. 1. When the animals were killed 2 h after *6* 15 o.osj: .1 U) J------ 1__ t 120 Time [hi 2t0 Fib 1. Level of SSB expressed as-log Fos fot DNA of (a) kidney; IhMiter; |c> lim*. (d) spleen, te) brain after different times of exposure to VC. SSB were determined after -M ) arid IS h <) after exposure, control animals ( J moan j S E Slalistical analyses were performed with the Student s Hew. The lowest tiadsitcat significance of the differ ! dCC *1* * n m e n v aJ u es ^ com r ots and o f ex posed anim als are give n k itlnc vs (39 h) p< 0 Oo. liver {137 h) *P< 0,0a. lungs (39 h) **p < 0.01; spleen (234 h) **P -- Q 01 an exposure period of 117 h. increased levels of SSB were found in lungs, liver, kidneys and possibly spleen, but not in brain. For kidneys and lungs a plateau was reached after 80 h and 120 h exposure, respectively* The level of SSB in liver, however, seemed to increase slightly throughout the 6Z.S900 S^U Jfi exposure period. A statistically .significant increase in SSB occurred in spleen only at 23-1 h. The level of SSB after 231 h of exposure was about the same for kidneys, lungs, and liver when the mice were killed 2 h after exposure. When tlie animals were killed 18 h after VC cxposuic, the SSB levels m kidneys, lungs and liver had relumed to normal values for animals exposed for 3G h. i Lowever, after 11-1 h and 231 h of exposure the SSB levels m kidneys, lungs and liver remained elevated even 18 h after termination o f ex po.su re. In brain tlie SSB level 18 h after V C exposure of 11 -1 h, was the only sample elevated, The SSB levels in brain at other exposure periods did not show a consistent pattern. DISCUSSION VC is rapidly metabolized to chloroelhylene oxide [5], which can be conjugated to glutathione or be transformed to chtoroacetaldehyde, which can be further metabolized [10]. The short-lived [ 5] epoxide is the most reactive metabolite [11] and binds to nucleophilic sites in macromolecules [5]. The mam product of the alkylation of DNA is ,V-7 (2-oxoethyl)guanme [51. The VC metabolites also alkylate proteins [12] in the tner, small intestine, kidneys, lungs and spleen, but not in the brain. The tissue protein binding was roughly correlated to the monooxygenase activity The binding to protein is o tunes higher in Siver than in kidneys, lungs, or spleen [ 12.13 J. A --log F^s value of 0.11. which was reached for liver after 234 h of exposure tn animals killed 2 h after termination of exposure, corresponds to about 0 5 SSB/10'S daltons (calculated from Refs. 6 and 9). Thus, after 4--S weeks of exposure, one SSB seems to be induced per every 2 DN'A molecules m liver, lungs and kidneys. For kidneys and lungs, the SSB levels reached a plateau. There are several possible explanations for the formation of the plateau, e.g. only a limited amount of SSB can be formed during the base excision repair process. Chloroelhytene oxide may also act as a cross-linking agent, and thus re stricts a further increase or SSB, A third possibility could be thatchloroethyiene oxide might, after a sufficiently long exposure time, induce ceil death, leading to a degradation of DNA. Finally, induced and repaired SSB may attain a steady state. This latter hypothesis could be further illuminated by combining VC exposure with administration of substances inhibiting excision repair. The observation that the SSB levels are normalized 18 h after the first 36 h of exposure, but not after longer exposures, indicates that the induced SSB can no longer be repaired after a sufficiently long induction time, which lies somewhere between 3G h and 114 h of exposure. The curves shown for the different organs in Fig. la--c suggest that this induction time may be different in various tissues. SSB are formed by base excision repair of alkylated bases, e.g. iV-7 and 17 jV-3-alkytguamne [ 14,15]. Tlie alkylation of O* in guanine, which is related to mutagenic and carcinogenic effects of chemicals [1G], is not repaired by ^ excision repair [17] and docs thus not lead to SSB. However, as alkylating r agents react to a higher degree with jV-7 than with O6, of guanine, an increase * in the SSB levels might at least tentatively be used for definition of critical * organs. However, much work has to be done to further illuminate this possibility. It has been shown that DNA fragmentation caused by a carcinogenic substance tn a particular organ is related to the carcinogenic effect. This has for example been shown for nitrosoamines and alkyl alkansulfonate [22,23]. In the present study the highest levels of SSB were reached in liver but DNA damage appeared earliest in lungs and kidneys. The spleen seems to be less sensitive than liver, lung and kidney, as judged from the lower SSB value in the spleen at the end of the experiment. The lung is the most sensitive organ for VC carcinogenicity in mouse [3,20]. How ever. although liver hemangiosarcomas are induced also in mouse [2,3], they are less common than hemangiosarcomas in other organs and they represent only a minor portion of all tumor types formed. Reticulum cel! sarcoma of the spleen was observed in mice in one study (3|. Kidney tumors have not been detected in NMRI mice, although VC induced tumors occur red in perinephral fat tissue [3]. However, kidney tumors occurred in Swiss mice in Maltoni s study [2], The absence of a difference in SSB between bmr. tissue o: \ C-exposed mice and control animals is in accordance with me obser, ations [13] that no radioactivity bound to brain DNA could be detected in \ C-exposed mice and that no brain tumors have been observed in experimental animals, Brain tumors have been found among occupationally exposed men [21-- 2-1 j. acknowledgements tt e thank Dr. Siv Osterman-Golkar for valuable discussions on the manu script. We are also indebted to Pia Hanserkers and Karl Sigvardsson for skillful technical assistance and to Eskll Hjort for drawing the figures. REFERENCES 1 I ARC (1979) Monograph* on the evaluation of the carcinogenic risk of chemicals to man, IARC, Lyon, 19, 377--436, 3 Mahom, C. (1975) The value of predictive experimental bio-assays in occupation.il and environmental carcinogenesis An example: Viny I chloride. Ambio. 4.16, 3 Holm berg. 9., Kronevj, T. and U'inell. M. (1976] The pathology of vinyl chloride exposed mice. Acta Vet. Scant!., 17. 326--342. 4 Piugge, J{. and S. (19771 Vinyl chloride metabolism. A review. Chemospherc, G. 309--3 23. 5 Osterman Golkar, S.. Hultmark, 0,, Segerhack. D., Calleman. C.J , Clothe, It.. Ehrenberg, L. and Wachtnieister. C,A. (1977) Alkjlation of DNA ant! proteins in mice exposed to vinyl chloride, Biochcm. Biophys, Hes. Common., 76. 239--266. 6 IVmiles. S, A,S. ami Orsen, I. (1983) Singlc-ilronrt breaks ica UNA of various organs of mice iiuluccd b\ styrene anti styrene ox idc. Cancer Lett., 21.9 -- 1 5. 7 AUnstrbm, G. and lirixon, K (1973) Radiation iminced strand breakage m DNA from mammalian celts, it mud separation In alkaline solution, Int J Radial. Bio] , 23. 285-289. S Valles. S.A.S. and Emon, K. 1198 15 Single strand breaks in DN A ut various organs o f m ic e i il d need b > in c tli y I m c ilia n esti 1 fon a te anti d iiu etb y]su I fo x id c del e rm i netl by the alkaline u nwinding technique. Carcinogenesis, 5,3] 9--323. 9 Kris on, K., anti Abnstjom. G (1979) Single-strand breaks nr DN A during repair of UV'-induced {lainage in normal innnan anti Xeroderma pigmentosum cells as deter mined by alkaline DNA unwinding and Isydroxvlapatile chromatography. MuCat, Res, 39. 257-271. 10 C r e e n, T, anti J I a t h w a y. D. X. (197 7) Tls e c h ein i s try anti b ioge nesis of (h e S-co n l.iin- jng metabolites of vine} cblonde in rats. Client -Biol. Interact., I 7, 13 7--150. 1 \ Huss&w. S, and Osierman-Golkar, S (1976 > Comment on the mutagenic effectiveness of tiny] chloride metabolites. Chetu.-Biol. Internet,, 12. 26 5--267, 12 Bolt, ff.M., Filser. J.C., Uib, R.J anti Otterwalder. II, (1980) Binding kinetics of vinyl chloride and vinyl bromide at very low doses, Arch, Toxicoi, Suppl., 3, 129 -- 142, 13 flerpnan, K. (19S2| Reaction of vinyl chloride with HN'A and D.VA of various mouse tissues m vivo. Arch. Toxicol.. 49. 117--129. 14 Hu a n g, P. H T. a nd 6 t ewar l, B V. (197 7) Di f Te ren ces in patterns of structural change b> rat liver DNA following administration of dimethylmtrosamine and methyl methanesulfonate. Cancer Res.. 37. 3796--3801 13 Snyder. R.D and Regan J.D, (1982 DNA repair in normal human and Xeroderma pigmentosum group A fibroblasts following treatment with various meihanesulfonate and the demonstration of a long patch lU.Y.-likei repair component. Carcinogenesis 3. 7-14. )fi O'Connor. P J. <19S1J Interaction of chemical carcinogen* with macromolecules. J. Cancer Res Clin. Oncol., 99. 167--166. 17 Olsson, M. and Lindahl. T. 119S01 Repair of a I k via ted DNA in Etch e rich io eoh J. Biot. Chem . 255. 10369-10571. 18 Swenberg, J.A., Peuold, G.L. and Harbach, P.R. (1976) in vitro DMA damage/ alkaline elution assay for predicting carcinogenic potential. Biochem Biopins Res. Commun., 72, 732--738. 19 Petiald.G L. and Swenberg, J.A (1976) Detection of DNA damage induced rn i-ito following exposure of rats to carcinogens, Cancer Res , 38, 1589--159 4. 20 Suiuki, Y. (1963) Neoplastic elleclcl vinyl chloride in mouse lung -- lower doses and short-term exposure. Environ. Res , 32,91--103, 21 Tabershaw, J R and Caffey, W.R. (1974) Mortality study of workers in the matin facture of vinyl chloride and its polymers J. Occup. Med , 16, 509--316 22 Monson, R,R , Peters, J.M. and Johnson, M N. (1974) Proportional mortality among vjnj t chloride workers Lancet, 17, 397--396, 23 Beck, P.D., Martin, J.F., Young. R.S., Creech, J,, Selikoff, I.J., Falk, K . U'alanabe, p,, Popper. H. and Thomas, L, (1976 I Vmvl chloride associated liver disease. Ann. Ini ern. Med.. 81,7 17--731. 24 Waxweiler, R J., Stringer. U-., Vagoner. J K, and Jones, J (19761 Neoplastic risk among workers exposed to vinyl chloride Ann. N.Y. Acad. Sci . 271. 40--46 Cancer Letters. 25 (198-1) 19--23 Elsevier Scientific Publishers Ireland Ltd. SPONTANEOUS POLYPOSIS IN THE SMALL INTESTINE'OF GEtlM-FREE AND CONVENTIONALIZED BALB/c MICE TAKfcO HI?.UTAN1*.TETSUZ0U YAM AMOTO*. AKlnA OZAKI*. TSUTOMU OOWADA`ami TOM OTA PI MlTSl!OKA`'b Vtuirriaf Physiology Laboratory, The Institute of Physical and Chemical Research, H"a)fo-*hi, Soitamo 351 and b Department of Biomedical Science. Faculty of Agriculture, Vnn-enity of Tokyo. Bunkyo-ku, Tokyo tl3 (Japan} (Received 26 July 1984) (Revised version received 29 August 198-1) f Accepted 30 August 1934) The spontaneous polyposis in the small intestine of germfree (Gf) and conventionalized (Cv) BALBc mice was studied. Gf mice were bred in our laboratory and maintained Gf in vinyl isolators. The first generation off spring of the Cv mice derived from the Gf mice was used as Cv animals. When they u ere 12 months old, the animals were killed under COj inhala tion and auiopsied carefully for the number and size of poJyps with the aid of a dissecting microscope. The incidence of polyposis was higher in the Gf mice (6S% in female and 89% in male) than in the Cv mice (37% in female and 51% m male). The number of polyps/mouse was also higher in the Gf mice (2.3 in female and 5.7 in male) than in the Cv mice (0.8 in female and 1,3 in male). All of the polyps were his to pathologic ally adeno matous and developed only m the upper part (mainly duodenum) of the small intestine. The present study demonstrated that development of poly posis in the small intestine of BALB/c mice was suppressed by the presence of intestinal microflora. INTRODUCTION Spontaneous tumors of the small and large intestines of mice are relatively rare, but adenomas and adenocarcinomas occur occasionally in all segments of either the large intestine or the small intestine of some strains of un treated mice, such as C57BL or BALB/c mice (6). The development of polyposis was unexpectedly noticed in the small intestine of untreated germfree mice in an experiment aiming .to study the effect of intestinal bacteria on chemical carcinogenesis in the intestine of gnotobiolic BALB/c 0304 3835/84/S03.00 1984 Elsevier Scientific Publithers Ireland I-lri. Published and Punted in Ireland L8S900 S^d