Document QkZbKmKYXkz4681DMKQydwNYE

i. .t 'I I .\furorin Rrrrorch. 135 I1984) 203-209 Elserier MTR 00861 Cytugenetic effectsof benzene: dosimetric studies on rats exposed to benzene vapour J.A. Styles and C.R.Richardson Inrprrtul Chrmtcul lndurmes PLC. C m m l Tu.ricolo,qvL u h ~ u u i pA, idrrlv Park. .Wucclex.firld CheshirerGreur Ertrotn) (Received 29 March 19831 t Revision received 28 July 1983) I Accepted 13 December 19831 Summine Rats were exposed to benzene vapour at nominal concentrations in air of 1. 10. 100 and lo00 ppm acutely for 6 h. Bone marrow cells from each animal were examined for chromosomal abnormalities 24 h after the end of the exposure period. This analysis was carried out on 150 metaphases per animal where possible and showed a significant increase in the percentage of cells with chromosomal abnormalities. excluding gaps. in the groups of animals exposed to 100 and lo00 ppm benzene. In the 10-oom and 1- +exposure groups there were elevated levels of cells with abnormalities which showed evidence of etng dose-related. although they were not statistically significant. Benzene is non-mutagenic in all of the point tion. Numerous cytogenetic studies have been done mutationd assay systems in which it has so far on bone-marrow cells or peripheral lymphocytes been tuted (Lyon. 1975: 1976: Jerina et al.. 1968: from human subjects exposed to benzene vapour Dean. 1978: Shahin and Fournier. 1978;Cotruvo (Dean. 1978;Pollini and Colombi. 1964a.b: Pollini et al.. 1978: Tanooka. 1977: Rosenkranz and et al.. 1964. 1969: Pollini and Bicaldi. 1976. 1977: .Leifer. 1980). It can induce chromosome damage Fomi et al.. 1971a.b: Forni and Moreo. 1967.1969: in plants and mammalian somatic cells in vitro Hartwich and Schwanitz. 1972: Sellyei and Kele- (Gomez-Arroyo and Villalobos-Pietrini. 1981: men. 1971: Erdogan and Aksoy. 1973: Hudak and Koizumi et ai.. 1974:Morimoto. 1976; D i u et al.. Gombosi. 1977: Van den Berghe et al.. 1979: I 1979: Morimoto and Wolff. 1980;Gemer Smidt and Friedrich. 1978). mammalian cells in vivo (Lyon. 1975: Zhong et al.. 1980: Diat et al.. 1980: Tough and Court-Brown. 1965: Tough et al.. 1970; Funm-Cravioto et ai.. 1977;Picciano. 1979: Khan and Khan. 1973: Frcdga et al.. 1979; Watanabe et Hite et al.. 1980; Siou et d.. 1980a: Meyne 3nd al. 1980).The cytogenetic investigations indicated Legator. 1980; Sou et al.. 1980b: Dean. 1969: that at high exposure levels benzene is il human Philip and Krogh Jensen. 1970: Lyapkalo. 1973; and animal clastogen but the available data from Dobrokhotov and Enikeev. 1977; Anderson and inhalation studies were insufficiently consistent to Richardson. 1979)and also been shown to induce establish a dose-response relationship and de- SCE (Tice et al.. 1979. 1981).In the chromosome termine whether the present TLV (current OSHA studies in animals benzene was administered by standard is the 8 h time weighted average of 10 i.p. injection with the exception of the investiga- ppm) was adequate. The investigation reported tions of Anderson and Richardson (1979)and Tice here was designed to examine the cytogenetic ef- et 31. (1979.1981)where exposure was by inhala- fects of a range of concentrations of benzene Ot63-1218/84/503.00 *: 1984 %evier !kience Publishers B.V. vapour on rat hone marrow cells following inhalation and to define concentrations of benzene vapour that would he non-claitogenic under conditions of acute exposure. Materids s%dmethods Bett:enc. The benzene (AnalaR) used in this study was supplied by BDH Chemicals Limited. Pooie. Dorset. Reupnu for slide processitig. The following reagents were used: Hank's basic salt solution obtained from Gibco Biocult. Paisley. CK: potas- sium chloride (AnalaR) from BDH Chemicals Limited. Poole. Dorset. UK: glacial acetic acid (AnalaR) and methanol (AnalaR) from Hophin and Williams. Chadwell heath. Esses. LK: col- chicine from Sigma (London)Chemical Company. Poole. Dorset. U.K. and Giemsa stain (Gurr's R66) from Searle Diagnostics. High Wyombe. Bucks. UK. Animals Male. Wistar-derived rats. 6-8 weeks old. were supplied from the specific pathogen-free colony at Aldericy Park. Cheshire. UK. The rats were ran- domly assigned 10 cages using a shuffle card . method and housed 4 per c a p . Animals were given food and water ad libitum except during : exposure periods. Male animals were used because . they appear to be more susceptible to the clasto- genic effects of benzene ihan females ( M e y e and Legator. 1980). Exposure in humans is prtdominantly in males. ' Exposure 10 ben:ene Groups of rats were separately exposed to atmospheres containing benzene at 0. 1. 10. 100 and loo0 ppm for a single 6-h period. Each group of animals at each exposure level consisted of 8 rats except for the controis ( 0 ppm) which contained 12 animals. The animals were exposed to clean dry air in whole body chambers (Gage. 1959). The air was supplied at a rate of 9 I/min during the exposure period. Animals were generally caged individually but some were housed in pairs. A rniosphere generation The test atfnospheres were generated h! direct injection o f henzcne liquid into the air w c i m entering the chamhers. The henzene was delivered from a Hamilton- Bonaduz gas-tight qringc ( Hamiiton. Bonaduz .4G. CM7402 Bonaduz. Switzerland) of the appropriate capacity at an injection rate governed hy a Sage Controlled Injection Pump. Model 355 (Sage Instruments Division. Orion Research Inc.. Cambridge. MA. U.S.A.). The injection rate was adjusted to bring Ihe concentration range within k 10%of the desired level. A ttiiospltere u t ~ u l r s ~ ~ Benzene at the lo00 ppm level was monitored using a Wilks Miran 1A infra red analyzer (Foxhoro Analytical. Milton Keynes. Bucks. U K ) and at the lower concentrations hy gas chmmlltopraphy after trapping the test substance on Poropak Q (80-100 mesh) (Waters Associates Inc.. Milford. MA. U.S.A.). Proccsstng oj rar hom niurronv Animals were killed 24 h after the end of ex- posure as recommended by the ad hoc committee report on mutation testing (1972). Legator et ai. (1973) and Sugiyama (1971). The bone-marrow samples were prepared according to the method of Sugiyama (1971) but with slight modifications. The method used was as follows: ( I ) Each rat received 3 mp colchicine/kg bodyeight intraperitoneally 2 h prior to sacrifice to arrest dividing ceils in c-metaphase. This was administered to each of the randomly housed groups working across the rack. This sampling order was maintained throughout further processing until fixation occurred. (2) The rats were killed by cervical dislocation. both femurs from each animal were removed and the bone marrow harvested by aspiration with Hank's basic salt solution. (3) Cells were treated with hypotonic solution (0.075 M potassium chloride at room temperature for 20 mini followed by fixation in glacial acetic acid and methanol in the ratio 1 :3. (4) Slides were prepared by air-drying and stained with Giemsa (Gurr's R66). ( 5 ) The slides were coded to avoid observer bias while being scored and were read in a random order which was computer-generated. 250 cells from each animal were examined where possible and any abnormality was assigned to one of the following categories: I i II stream iclivered syringe madut. at an :d InjccIivision. U.S.A.). he cnn=.dlevel. mi tored (FOX- K ) and iatogra'oropah 4 i l ford. of exnmiltee r et ai. epared 71) but was as ine/!kg icrifice is was loused npling 'OCCSS- killed each arrow c salt otonic room ion in ) 1:3. iained were cored \ was nimai rmalones: TABLE 1 G R O U P MEAN PERCESTAGE OF CELLS WITH ABSOR%l.4LlTIE!! Acute 6-h erpmure. 24 h kill @ xaw n a l i t i a Dose le\el or hcnzcnc cppmi 0 Contrnl 1 IO IOU -- - 1 (K)(l Including gaps Excluding g p s ! 1S 4 0.25 (12) 2.00 0.41 (PI 3.03 0.9 1 (8) 445 *= 1.23 ** In) 781 ** 4 49 - 9 (8) The number in hrrckets 15 the numher of animals which were eramind. go Statistically SignifiC;mtlygreater than the control group mean at the 1% level 1 t-test. one-wded on tran&rrned data, ( a )chromatid or chromosome gaps: ( b )chromatid breaks: (c) fragments: (d) minutes: (e) any other complex abnormalit? such as Rohertsonian translocations. The criteria for the above classification is given by Anderson and Richardson (1981). analysis allowed for the 3 different cytogrnetici\ 6 who examined the slides. The group means &ere adjusted for reader differences (and therefore d 3 not correspond to a straight arithmetic mean c f the data J before the mean transformed proportiori for each benzene group was compared uith t h e Slaristicai anairsis The proportion of cells with any abnormalities and the proportion of cells with any abnormalities mean transformed proportion in the control group using a one-sided Student's mest. The test u a , based on the error mean square in the anal>s!c of variance. i other than gaps were transformed to stabilise the variance using the double Arcsine transformation Results 1 i (Freeman and Tukey. 1950). The transformed data Test atntosphee ana!rsis ! were then considered by analysis of variance. The The test atmospheres generated uere: 0.8 5 0.1 ! TABLE 2 ,MEAN PERCESTAGE ISCIDESCE( +SD, OF CELLS WITH DIFFEREYT CATEGORIES OF CHROW)S0M4L D 4 I l A G t FOLLOWING EXPOSCRE TO DIFFERING ATMOSPHERIC COSCESTR4TIOXS O F BENZESE i Group Number N u m k r Mean Mean Mean %lean h4c3n Ii Of of cell5 !f cells with Q cells with S cells with P cells uith F cell5 u i t h animals analysed chromosome or chromatid chromosome hrerhs minuter inicruhiinp I control I2 pet group 2641 chromatid gaps 1.40 2 1.22 breaks 0.06 20.13 or fragments 0.34 f0.80 0.08 ,+O.lR 0 I 1 PPm 8 ! benzene 1 846 1.88 5 0.72 0.0s 20.15 0.37 0.411 00 I 10 PPm 8 benzene 1193 1.bR 2 1.48 O.OR 50.24 0.34 ,+0.51 *0.17 0.48 O.OR -0.24 1OOppm ' benzene 8 =1429 3.16 2.40 0.21 +, 0.29 0.49 0.76 0.14 2 0.26 0 1OOOppm benzene 8 2039 2.40 2 1.03 0.15 2 0.20 3s 2 1.66 0.20 ~0.55 0.10 =O.lR Diwwsion imals exposed to loo0 pprn i immediately after the exfrom other exposure levels rrcentage of cells with in Table 1. There was a crease in the percentage 5. including or excluding to 100 and loo0 pprn s of exposure there was the percentage of cells -malities. There were 31 cells which showed ted in the 1-ppm and t these increases were :idence of cells with mosomal damage is Ice of different cateven in Table 3. Human exposure to benzene is predominantly hy inhalation. hence the results of this study sup gest that an acute exposure level not exceeding 10 ppm isynlikeiv to cause ctastogeniceffects in man provided that rat and man are phannacodgnamically similar with respect to benzene. This conclusion is supported hy data from chronic exposure studies in humans where concentrations of benzene were estimated. Forni ct a]. (1971a.b) obsewed chromosome abnormalities in lymphocytes from p p l e e x p e d to 125-532 ppm benzene: Tough et al. (1965.1970) found that exposure of workers to 25-150 ppm benzene caused a signifi- cant increase only in the unstable aberrations in lymphocytes whereas lewis of 12 ppm did not induce any significant increase in chromosomal abnormalities. Picciano (1979) has reported that in subjects exposed to < 10 ppm benzene there was an increase in the incidence of certain categories of chromosomal damage compared with controls. al- though the percentage of abmanr cclts was not significantly different. This study has bem criticised by Dabncy (1981) on the following grounds: first. there was inadequate information on extra- .L!j WITH DIFFEREXT CATEGOR~ESOF CHROMOSOMAL DAMAGE PER CELL :RING ATMOSPHERIC COSCE?c'TRAflONSOF BESZESE an dence of momme or mrtid gaps cIlx10'~ Man incidenceof chmmriid bmk5 p*.cellx10-' 0.038 f0.13 0.054 20.16 0.034 f0.23 0.21 f0.28 0.147 f0.20 Mean inndmcc of chromosome bmkr of f r a ~ l S prrCellXtO'* *0 . 3 ~ 0.89 Mean incidenceof minures prrCdfK10-' 0.076 +0.1R 0.37 i0.49 0 0.42 i0.61 0.m 2 0.47 0.49 ?:0.77 0.14 k0.25 4.12 f 1.94 0.29 20.m Mean incidenceof interchanges pClCCllX10-' 0 0 0.084 k0.24 0 0.147 f0.26 - s t u d i o in workerm exposed 10hmtrnc IW rnlurne or h h . Arch. Environ. Health. 22. 373-375. Frcdp. K.. J. Rcicalu and M. Berlin (19791Chrt~mcw*mr studies in workers erpcwrd IO h m r ~ n c .In: Gcncric Damage in Man Caused h? Entimnmtal Agents. Academic Prcm. New Yo&. p. 1R7. Freeman. M.F.. andcJrw. 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