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t c Jerrold M. Ward, DVM. P h D John H.Weisburger. PhD; Richard S. Yamamoto, ScD Timothy Benjamin: Carolyn A. Broan, MS; Eliubeth K. Weisburger, PhD Woanllng ma10 CSISL/6N mko re- mice was used to test the potential ef- once weekly until 54 weeiu. Yice given 3U% d v e d repeated s u t ~ u t ~ I~nja~ctulorns d knrono In corn dl for 54 woeks. Thoso mlto and sudving contmlsmn UlM 104 *rooks anw the flml Inl.ctlon. There was no widuua ot urrlnogmIc activity In bonzenclnm1.d mlt.. A high Incldenca of amyloldorlr dml0p.d in mlw of all b8at.d and untnatod groupr. B~~tyinltroroureau.sed as a po~ltlwcon- -, Inducod lymphomas or I.ukOml.8 (In fect of benzene and develop an animal model."." Whereas the normal route of exposure in man is by inhalation or by cutaneous absorption, we elected to develop the model by testing benzene by subcutaneous injeo tion, mainly in order to administer as high a dosage as possible. If positive results were obtained, then other benzene rere iajected with 0.05, 0.5 0.1. snd 0 2 mi for the fint four weeks. respectively, followed by 0.2 ml until the last injectma. Oil-injected mice received 0.025, 0.06.0.05, and 0.1 mi. respecuveiy, far the h t four weeks followed by 0.1 ml until the lut injection The lower dose given to the &injected mice was used when i t n s f m n d that 0 2 ml of corn oil injected twice m k l y did not disperse rapidly enough. ~ T o % p p - a a - m I u i ~ - j ~ ~mOuteGs simulating more closely the However, mice receiving 3096 k w n e in In a few. human route could be used. corn oil readily absorbed the 0 2 ml injec- tion,of which 0.14 mi consisted of corn oil. Many reports suggest that in man industrial exposure to benzene MATERIALS AND METHODS Weanling male C578L/6N mice were o b At 104 wceks after the fint injection, all lmrviving mice were kiIled ( X O 8 reeks of age) a d a complete necropsy vu per- increases the risk of leukemia.'-' t.ined from the Animal Production Sac- formed. LI had been done with all mice However, attempts to induce ne+ tion. Division of Research R ~ r a s , that died T k u e s were hed in neutral plaams in laboratory animals have National Institutes of H u i t h . They were buffered formalin. embedded in purffln, generally failed.'." Since C57BL mice have been shown to be quite sensitive to agents like radiation or chemicals that induce leukemia. this strain of housed. tea to a polycarbonate age Kctioned at 6 p and stained with hema(29x 17x 1w cm) in an ailcconditioned an- taxylineoain S d d tissues were stained imal mom. Food was loboratory chow with Congo red for amyloid and by the (Wayne Lab Blox) and hardwood chip - riodic reid-schiff method PortiorH of liver were d for bedding. After one week of fromone mousewith amyloidosi. were ked acclimatization, the mice were injected in gluturldehyde for ultrutructural ex- Sobatitted for publication July 25. 197(; .c. aptcd hull Prom th. Cucinogw Yet.bdism and Tox- icdogy B m c h (Dr.Ward. Mr. Benjamin. Mr Bmm. And Dr. E Weisburger)and the Expenw a t d Patholm Bruvh (Dr.Yunamoto). N.- Ll0a.l CUI=? Iaat~tut.. &tbcd& Yd. md the subcutaneously with benzene (80 mice) or corn oil (20 mice) or were untreated (20 mice). The benzene (99% pure) for injection m a prepared aa a 30% solution by meaauring 30 ml of benzene and diluting i t with liquid corn oil (Mazola).to a find volume of amination, and processed for atudy in an dceLron m#roscope. 16 8 positive control, butyiaitmeourea (mdtingpoint 82 C). synthesized d i n g to tbc procedure of Werner," w u given to aide C57BL/6N mice, 4 weeks ofage, in American H d t b Fwndatian, New Yo* (Dr. J. Warburger). Repnnt rtquwu to BIdg 37. R m m 3827. National Cancer Inruwre, HIH. Bothesda, M D Zoo14 (Dr. E. Wciabuwr). 100 mi. After several toxicity &ah, the injection schedule developed for this experiment w m as follows: mice received injections twice weekly for 44 weeks, and then the drinking water a t a concentntion of 1 mgi3 ml (approximate daily dme) of water for 120~ Y S S. urviving mice were killed at 52 weeks. 22 Arch Ennron HealthNd 30. Jan 1975 -. Long-Tsnn Effect of Eenrener Ward et al sensitive s i t s - R_elease-or activ-at-ion lit aleukomogenic virus also deserves consideration. The lymphomas induced by butylnitrosourea were identical to those reported previously in mice.'' This is the first report of the finding of intestinal tumors in mice, although they were found previously igsome rats given the c h e m i d l * ~ a s The development of intestinal n e plasms is similar to the recorded finding of such tumors after oral dosage of methylnitrosouma.'. I t would a p pear that the fairly polar chemical is not atworbed readily, migrates down into the lower intestine, and thus leads to carcinogenesis along the entire tract. The finding of mites on the chronic skin lesions is reminiscent of previous reports on the subject*'-n Evidently, the scratching caused by the mites leads to acute skin lesions. In more recent experiments, with C57BL/6N mice, the scratching be gins a t 7 to 9 months of age in mice kept on hardwood chips only. Cedar bedding or mixtures containing cedar chips had both a preventive and curative effect. For an unknown reason, the black mice were more susceptible to thin condition than were other strains. The chronic skin lesions that developed led to amyloidosis in internal organs and eventual death.z7.'n.u The mean survival of these mice would probably have been increased if mitea were not present or if cedar bedding had been used." However, benzene exposure did not appear to affect this disease procesa Supporccd in puL by o D n m NIH 73-G M1CC to B P e r H i m L.&Grcat FalL. VI h m a Lam Ambmc. PIUICU ut. William, Jun Kelle?. and Ameli8 C r r a t aided in lhir -dY. Rderenca8 1. Aksq MKN. Akgun T. Erdem S. et .I: Remalologid eff- of chmnic benzcne poirob inR in 217 d e n . Br 3 I d Mod 28B6402, 1971. 2 Bmwaiag E Torieity and Metabdiam ojln- d u t r i d Sdwntr. Amnerdam. Elsevier Pub1i.h- iag CO. 1365. pp 3-66. 3. Coldwater U:D i s t n h n w in the blood following expaurn to benzene. J Lob Clia M d ~957-961,1941. 4. de Gowin RL: Benrcne exporum and apl- tie anemia followed by leukemia five.yean later. JAMA 185:74%751.196a. 5. Iahimun T.0- A.Tomiyuu T. e t .I:& mpatiowl facto- in tb.e p i d e m i o l w of leuke- mia in l i b h i m a and N W A m J Epi- h i d 95l57-166.1m 6. Road.odii EG.Gorini P.GenuG. et ak Pa- thoiogy of erythroblutic m i b in oa~upatiolul bcarcnw uythmpstby and erythremia. Eibl ? I d 3&1-18&1970. 7. T d EM, Xonchialoniuya NM. him LA: Benzene leukemia. Acto Ua I a t Ca- 195'51-755, 196S. a Vigiiani EC. h i t . G: Benzene and leakc m i r N E y l 3 Xed 'Zl1572876.1964. 9. Amid fiN-tive attempt at induction of leukemia in mica by benrcnc Rw Franc C L d u Qir Bid &198-199,1960. 10. Ivenen OH.lvenran U A itudy of epider- mal tumonrigenak in the hairlcu mouse with mgle d repeated applicatlonr of bmethyi- ehoianthrcne at different Act8 Mieohd Scad 6ZUlS314.1964. 11. Kirrhbaum & Stmng LC: Influene of cudnogem on the age ineidenoc of leukemia in tbc hi& leukemia strain F of m i a COM Ru 2241-845.1912. l!L h r u m OD: Reticulum 4 1 aeopiumr in normal and b n u n a tmrsd hurl- miak Acro AtAd Micro6id h a d (A) 815743,1913. IS. K s p h HS, B m r o Y B A quanti- b r e s p o n r ntudy of lympboid-tumor develop. meat in irradiated C37 Black m i a J Notl Canm I H L 13185.208.1952 14. Yokom K. I m l m u n N.Takiura S, et d: Lcukemogrnie and m a m m tumorigenic effectm of N-nitroaobutylwa in mica and mt& Gaaa 613287-289.1970. 15. Werner EA: The rnnstitntion of a r b mid- P u t IX. Tbe internetion of nitmua d d and monctwbstituted we.* The preparation of di.oDmctbane. diuocth.ne. diax+n-buunc. and diamiropmtana fnrm the respective nivct loarea& J C k m sa 115109bllM 1919. 16. lhrnn TB, Deringer M E Reticulum all neoplum. t y p B.orthe "Hodgkin'slib Laion" of the m a a n J Natl Camcu I d ron?l-8u. 1968. 17. Dunn TB.Normal and pathologicu u t o m j of the reticulu tiasue in Iaborator~mi- J Na!i C a w I- 14:L2Bl-llS3.1954. 18. Mamuhit. T:Expenmental d i a on tbe dinturban- of hematopoietic o w due to kn- zene intoxication. Nagoya J ,Wed .Sa 2lU04-234. 1%. 19. Nocske K. Y U ~ MH: AindMd and hiatometrid investigations of benslne-indued lcsionr of mt bone m m . Vitclunva A d [Po, thd A d ] 355504.1972 20. Philip P. Jenaen M K Benzene induced chromosome abnormalities in nt bone m v m r alla A d o Etrlhd M i o b i d S a d ( A ) 78:489-490. 1970. 21. Ddchnumn WB. MseDodd W E B u n d ETbe bemopoietic tiwuc toxicity of benzene OL. p ~-r'r2 A H P k d 6aO1-2M,1969. P Yah 5: Skin in h n i e uaenieiam. Hum Arud 4:469185.1973. m. C n w a PL. Hewer A S i m P Formation of K-region epoxides u micmwmd metabohm of pyrenem d b e a m t c p p n e . Bimckm plu- d 212713-2726.1972 24. +bi& H. Day JW. Jerina DM: Amne oxidor am rntcnnediatcr in the oxidative m e 6 lirm of aromatic mupoudc Iromeriutioa of meth+sub.titutcd u m e o x i d a Bidem&fy 1- ,1972 25. Oduhima 5: L.ulremogenemia of N-ai- IDbltJlpIu i n th n L Gaia 61245-25?l,1970. 26. Lu~aDD.S-n PF. M a g e e PN: Tbe in- duction of tumoun in tbe rat by a rinple o n l d o u of N - n i t m a m e t h y i u r u Br 3 C a m 253131-181,1969. 27. Dum T B A m y l o i d a i in mi&. in Cotrhin E, Roe FJ (e&): POtMqyof Z+atory Rata and M i a . Phikdeiphia, FA D.N Co, 1967, pp 181-212 28. Cdton Y: Mpobic mange in the molrra leading to akin ulacratioa and . m y l o i d a i r Am 3 FbcM 43:85586z1963. 29. Baton WE,Dcringer MK: H e r e d i t y re- n d divrw and amyloidoai8 in mia. Arch htAd MdeSB.Is4a. 30. "bung PJ: Senile a m y l o i d 4 i in mi-. Gcr- OnLdOpro 1269279,1457. 31. StQcL1J B Longevity and grosn pathology at death in P inbred m o u r rtninr J G e m k i n:UM409.196& I '. Arch Envirun Health/Vol30. Jan 1975 Long-Term Effect of Benzene/Ward et a1 25 .