Document QJRBeY2gkyDqLoj5ZKebZEekE

.vuicltionResearch, 77 ( 1 9 8 0 ) 149- 155 ,TEkevierlNorth-Holland Biomedical Press 149 ,l-HI< EFFECT OF BENZENE IN THE MICRONUCLEUS TEST ~ v I I - \ RHKITE, MARIE PECHARO, INEZ SMITH and SUSAN THORNTON Department o f Safety Assessment, Merck Institute f o r Therapeutic Research, Merck Sharp und Dohme Research Laboratories, West Point, PA I9486 (U.S.A.) (Received 25 April 1979) ( R ~ ,Vion received 17 August 1 9 7 9 ) O ~ C ' x e d 3 October 1979) Summary The potential cytogenetic effect of benzene was studied by means of the micronucleus test in developing erythrocytes of male and female mice at dosages ranging from 0.0625 t o 2.0 ml/kg/day. Groups of mice were given two dai? ora! doses and sacrificed 6, 1 8 , 2 4 or 48 h, or 5, 9 or 16 days after the last dot T!:e doses were given 24 h apart. Bone marrow from the mice was processzd for visualization of micronuclei in polychromatic erythrocytes. The results showed a significant ( P < 0.05) increases in the numbers of poly- chromatic erythrocytes with micronuclei from mice sacrificed 6 h after being given dosages of 0.25 ml/kg/day or more of benzene. Similarly, significant ( P < 0.05) increases in polychromatic erythrocytes with micronuclei were seen in mice given 0.125 ml/kg/day or more of benzene and sacrificed 18 or 24 h after thti second dose. However, in a second experiment negative results were ob: ,led at 24 h from mice at 0.0625, 0.125 and 0.25 ml/kg. One group of mi( (0.25 ml/kg/day and killed at 48 h ) showed significant increases in the numbers of polychromatic erythrocytes with micronuclei. Mice killed 5 days after treatment with 2 doses of 0.125 ml/kg or more of benzene showed significant numbers of micronuclei in polychromatic erythrocytes. The groups of mice at 0.5, 1.0 or 2.0 ml/kg/day and killed 9 or 1 6 days after the second dose of benzene showed values that were comparable to the corresponding Wc.tive controls. In all cases there were sufficient numbers of cells to analyze. A i >ughbenzene is a kqown hematopoietic poison and bone-marrow depres- there were sufficient numbers of cells t o evaluate in the micronucleus tes Thus, these experiments uphold the usefulness of the micronucleus test as a screening procedure in cytogenetics. During the development of the micronucleus test for inclusion in a shortt e r : ~carcinogenic/mutagenic testing program, several reference compounds I I I i i i i\ I/ t ! I i I Ii I I 1.50 I '",E;o,l I 3 I zzzl I I I I 7-1 w' cl -L9f. E a !i Y 5 E Y F ae a d dN :;were employed in our laboratoq - and hematopoietic poison [2,7,1 years, evidence has accumulated mias in industrial workers wheri information from clinical and ep for Occupational Safety and Hea genic agent [13]. The reason fc malignant disease of the blood. mends that for regulatory purl humans. The Consumer Producl tional Safety and Health Admin in the Federal Register [5,14]. OSHA's conclusions that emplc hazard, specifically the hazard of The micronucleus test is basc matin in the cytoplasm of you] previously mentioned benzene seemed worthwhile to investigat ity in this test. Materials and methods The potential cytogenetic efi micronucleus test in developing t experiments as outlined in Tab11 procedure of Schmid [14,153 wi The mice used in the various strain. They were about 5 wee animals were maintained in sta ditioned room (about 22C) wi water at all times. The benzene used in these e: tific). It was diluted (1: 5) in an Raynard, Inc.). The solution N glass syringes and stainless stee mice received an equivolumetric In 3 experiments, a positive was used. This compound was 1 solution (2.6 mg/ ml) at a doz benzene-treated animals. The c starting at approx. one-third of kg or 5.35 ml/kg, for mice [31.' The mice were sacrificed by days after the second dose. BOI Prepared for microscopic analy row from each mouse was evac centrifuge tube, using fetal-ca 5min at 1000 rpm. The super c*' N I aslo 151 tvctreemployed in our laboratory. Benzene, a known bone-marrow depressant a I ~ dhematopoietic poison [2,7,17], was one of these compounds. In recent years, evidence has accumulated implicating benzene in the etiology of leukemlas in industrial workers where benzene was used heavily [4,9,12,18]. This from clinical and epidemiologic studies led the National Institute fat. Orcupational Safety and Health (NIOSH) to identify benzene as a leukemo- IC agent [131. The reason for this is that the chemical causes progressive, m I lgnant disease of the blood-forming organs, and as such, NIOSH recommends that for regulatory purposes benzene be considered carcinogenic in humans. The Consumer Product Safety Commission (CPSC) and the Occupatwnal Safety and Health Administration (OSHA) published benzene standards in the Federal Register [5,14]. The basis for the standards were CPSC's and OSHA's conclusions that employee exposure to benzene presents a cancer ha:ard, specifically the hazard of developing leukemia. -'ne micronucleus test is based on the detection of small particles of chromL m the cytoplasm of young (24-30 h old) erythrocytes [8,15,16]. As prmiously mentioned benzene is a known bone-marrow depressant, thus it seemed worthwhile to investigate a chemical with this type of biological activity in this test. Materials and methods The potential cytogenetic effect of benzene WES studied by means of the IT. ronucleus test in developing erythrocytes of male and female mice in several e2c;eriments as outlined in Table 1.Each test was done following the reported procedure of Schmid [14,151 with minor modifications. The mice used in the various experiments were of the Charles River (CD-1) strain. They were about 5 weeks old and weighed between 1 0 and 20 g. The animals were maintained in standard clear plastic mouse boxes in an air-conditioned room (about 22C) with free access to food (Purina Lab Chow) and h & x rat all times. 'he benzene used in these experiments was of reagent grade (Fisher Scienti. 2 ) . ir;was diluted (1: 5) in an edible grade of peanut oil (Magnus, Mahee and Raynard, Inc.). The solution was given to the mice by oral intubation using glass syringes and stainless steel gavage tubes. The negative control groups of mice received an equivolumetic amount of peanut oil (10 ml/kg). In 3 experiments, a positive control agent, methyl methanesulfonate (MMS) was used. This compound was injected intraperitoneally in mice as an aqueous solution (2.6 mg/ ml) at a dose of 90 mg/kg under a regimen similar to the i- zene-treated animgs. The dosages of benzene were chosen by arbitrarily ting at approx. one-third of the reported oral LD,, value, which is 4700 mg/ li- or 5.35 ml/kg, for mice [31. The doses were given 24 h apart. The mice were sacrificed by cervical dislocation 6, 18, 24 or 48 h, or 9 or 1 6 days after the second dose. Bone marrow from both femora was harvested and Prepared for microscopic analysis after the method of Schmid [151. The marrow from each mouse was evacuated from the marrow canal into a siliconized (' siltrifuge tube, using fetal-calf serum, and the tubes were centrifuged for 1- nin at 1000 rpm. The supernatant was removed with Pasteur pipettes, and i 7a r a ;x -?1 00, I " , 3- 31 a ".? 1'9 "N. I "0,.0,"% I I m w oh h h h f' 9 90! 3 xI * Po P%A mo 2 ;;* E * oc I c?? ? ? ?c? 0 ~ 0 0 , " sI I I 2; 0 h as 1 R $ $h h h mwm .- h : A x :i0 2w, 3 m wI 9eu. 0% I I c ? t -"9 "eu. 3ZN33% I I w -0 9 m a wh -$ h h h 7 ; "MU0 ? l? 1 ;;9> N 00, 1 'o9"v. Foeum. w rI ?-" 00,00,0% I I I 0 2h N E0 3 ~ 5m z I uE zs ga z o 0 , I: ";mh -o hd ". 7 -'w0l 4a= -m10"I ,a*?s43 I I I t.30 2 s :m a 'E1 3zaV z 2 g % 02: h "m + I om '0901, I h - mmS '??a? 7 7-7 0 5m a w3 ffim t-3 qN Wo OCOO, : >2 .x z Z%S 5" 4 h N ? 0 w Ir "0 ah 2 %i p-4 22p 00, II 2 w -wA 3h m2 1-7 w m wffi N.9 "@! N.N. I mSNO_mz d a? ,"$ Nd * E Em b 8 a h *9 !Y **E II 0 .*0de 0Z d 1 0c E0 \I 5Y 5 2: %c t i c0 -k E; e$ .- .0- #0 : bt 'EEE", 2; @4 .E li-0 5Ejg ':5$ % ' .wE op *b aa cs 0o 8g :s g E; s using a rankit transformation I Difference procedure for p&V a treated group [7,171. Results 1 The results of the various e: represent averages and range ' erythrocytes with micronucle cell type from both male and responses were similar in ma seen in normochromatic ery' meAnltl. mice appeared in n o m I' sign of toxicity. Furthermo sufficient numbers of cells to ' The data show a significan matic erythrocytes with micl ' dosages of 0.25 ml/kg/day or increases in polychromatic f given 0.125 or 0.25 ml/kg/d dose. Two separate experimenh doses of benzene at 0.0625, 0.05) increases in the numb1 compared to the corresponc doses 0.125, 0.25 and 0.5 micronuclei in the same type With the exception of t k 1 significant (P< 0.05) increz with micronuclei were seer (0.0625 and 0.125 ml/kg/da Mice killed 5 days after 1 or 0.5 ml/kg) showed signi chromatic erythrocytes wit1 The groups of mice at 0.' 153 t1:e cells then were resuspended by mixing gently with siliconized pipettes in the small amount of serum remaining in the capillary part of the test tube. A drop of the cell suspension was placed on a clean microscope slide and spread In the conventional manner. 4 slides were prepared for each mouse. The smears were airdried on a slide dryer at 37C and dipped briefly into absolute n.dhanol for fixation. The slides were stained with May-Gruenwald Giemsa ,in.The microscope slides were coded so that they could be read in a blind al d random fashion. Approx. 3000 polychromatic and normochromatic erythrocytes from each mouse were examined for the presence of micronuclei. Microscopic evaluation was done with brightfield oil immersion optics at 1250X. The statistical analysis consisted of (a) the analysis of variance procedure using a rankit transformation of the data and (b) Fischer's Least Significant Difference procedure for pairwise comparison between the negative control and : reated group [7,171. Results The results of the various experiments are summarized in Table 2. The values represent averages and ranges of the frequency (per cent) of polychromatic erythrocytes with micronuclei compared t o the total scored population of this cell type from both male and female mice in the various treatment groups. The - q o n s e s were similar in male and female mice. The number of micronuclei er. in nonnochromatic erythrocytes did not show any relationship to treatzient. All mice appeared in normal physical conditions and none showed any overt sign of toxicity. Furthermore, when the smears were examined there were sufficient numbers of cells t o analyze from mice in all groups. The data show a significant (P< 0.05) increase in the numbers of polychro- matic erythrocytes with micronuclei from mice sacrificed 6 h after being given osages of 0.25 ml/kg/day or more of benzene. Similarly, significant (P< 0.05) cr2ases in polychromatic erythrocytes with micronuclei were seen in mice ,!veil 0.125 or 0.25 ml/kg/day of benzene and sacrificed 18 h after the second dose. Two separate experiments were done at 24 h. In the first, mice given 2 daily doses of benzene at 0.0625, 0.125 and 0.25 ml/kg showed no significant (P< 0.05) increases in the number of polychromatic erythrocytes with micronuclei compared to the corresponding negative control, In the second test, 2 daily ioses 0.125, 0.25 and 0.5 ml/kg produced significant ( P < 0.05) numbers of iicronuclei in the same type of cell. With the exception of the mice at 0.25 ml/kg/day and killed at 48 h, no xgnificant (P< 0.05) increases in the numbers of polychromatic erythrocytes with micronuclei were seen in the two other groups of benzene-treated mice (0.0625 and 0.125 ml/kg/day) sacrificed at this time period. Mice killed 5 days after treatment with 2 oral doses of benzene (0.125, 0.25 or 0.5 ml/kg) showed significant ( P <0.05) increases in the numbers of poly- :hromatic erythrocytes with micronuclei. The groups of mice at 0.5,l.O or 2.0 ml/kg/day and killed 9 or 16 days after 154 the second dose benzene showed values that were comparable to the cone ponding negative controls. Discussion Several compounds of varying chemical classes have been reported to increase the number of polychromatic erythrocytes with micronuclei in test animals [16,19]. Many of these agents are considered t o be carcinogenic and have given positive results in studies with mammalian and submammaliar systems 1193. This should be contrasted with a series of 1 6 carcinogens i which 11gave no response in the micronucleus test in the rats [181. As reported by Lyon [ l o ] rats with 0.05 and 0.25 mg/kg of benzene on twl successive days showed significantly increased numbers of micronuclei than corresponding controls. A dosage of 0.025 mg/kg for two days did not cause any noticeable change. Furthermore, benzene has been shown t o cause chromosome aberrations in rats and rabbits [6] and in humans, both in vivo and in vitro [6,203. The effects of benzene in the experiments reported herein were dose-related within the expected variability of the test. The results of this study, therefore support information about the biological activity of benzene since it is reportec to be leukemogenic and clastogenic. Although benzene is a known hematopoietic poison, there were sufficient numbers of cells in the bone marrow of treated mice to evaluate in the micronucleus test. These experiments uphold the usefulness of the micronucleus test as a screening procedure in cytogenetics. Of course, if the animals were dosed with benzene for several days, then the cell population in the bone marrow conceivably could be reduced to an unsatisfactory level for scoring in the micronucleus test. 7 Harter. H.L.. Expected values 8 Heddle, J.A.. and A.V. Carrai yirradiation: Evidence that I 44 (1977)63-69. g Infante. P.F., J.K.Wagoner, (1977)76-78. 10 Lyon. J.P.. Mutagenicity stud $11 Maltoni. C., Research "bombs 12 National Institute for OCCUP I 3 Revised recommendation for I. 13 Occupational Health and Sa benzene, Federal Register 42 14 Schmid, W., Chemical mutag $ (1973)77-85. 15 Schmid. W., The micronucli Chemical Mutagens - Princii \ pp. 31-53. I 16 Snedecor, G.W.. and W.G. CI Iowa, 1967,p. 272. 17 Snyder. R., E.W. Lee, J.J. actions of benzene, Life Sci.. i 18 Trzos. R.J.. G.L. Petzold, M 1 t h e rat using the micronucleu ' 19 Vigliani. E.C.. and A. Forni. I 20 Wild. D.. Cytogenetic effecr micronucleus test, Mutation 21 Wolman, S.R., Cytologic an (1977)63-68. Acknowledgement The authors wish to acknowledge the statistical assistance of Dr. N.R. Bohidar and the technical encouragement of Dr. H.M. Peck, both of the Merck Sharp and Dohme Research Laboratories, West Point, PA. In addition, the authors express their appreciation to Mrs. Thelma Demetrius for secretaria; assistance. References 1 Bruckner, J.V.. and R.G. Peterson, Toxicology of aliphatic and aromatic hydrocarbons, chapter 9:in National Institute on Drug Abuse Research Monograph Series 15, C.W. Sharp and M.L. Brehm (Eds.). Review of Inhalants: Euphoria and Dysfunction. 1977,pp. 124-163. 2 Christensen. H.E., and T.T. Lupinbyhl, Registry of toxic effects of chemical substances, National Institute for Occupational Safety and Health, US. Dept. of Health. Education, and Welfare, Rockville, MD. 1975,p. 163. 3 Cohen, H.S.. M.F. Freedman and B.D. Goldstein. The problem of benzene in our environment: C l i t ~ cal and molecular considerations, Am. J. Med. Sci.. 275 (1978)125-136. 4 Consumer Product Safety Commission, Benzene, Federal Register 43 (1978). part IV,21838-21854. 5 Dean, B.J.. Genetic toxicology of benzene. toluene, xylenes and phenols, Mutation Res.. 47 (1978) 75-97. 6 Goldstein. B.D.. Hematotoxicity in humans, J. Toxicol. Environ. Health, Suppl. 2 (1977)69-105. i nparable to the corre we been reported to th micronuclei in te ;o be carcinogenic and .n and submammalian i of 16 carcinogens in 3 rats [MI. ;/kg of benzene on two 's of micronuclei than wo days did not cause iown t o cause chromoIS, both in vivo and in erein were dose-related If this study, therefore, :ene since it is reported , there were sufficient ) evaluate in the microf the micronucleus test ,he animals were dosed n in the bone marrow lvel for scoring in the 155 7 Harter. H.L.. Expected values of normal order statistics, Biometrika. 48 (1960)151-165. 8 Heddle. J.A., and A.V. Carrano, The DNA content of micronuclei induced in mouse bone marrow by y-irradiation: Evidence that micronuclei arise from acentric chromosomal fragments, Mutation Res., 44 (1977)63-69. y Infante, P.F.. J.K. Wagoner, R.A. Rinsky and R.J. Young, Leukemia in benzene workers, Lancet, 2 (1977)76-78. 10 -.yon,J.P.. Mutagenicity studies with benzene, Ph. D. thesis, University of California, 1975. 1 1 laltoni. C.. Research "bombshell" hits benzene. as reported in Chem. Week, 121 (1977)33. 1 2 -iational Institute for Occupational Safety and Health, U.S. Dept. of Health, Education and Welfare, Revised recommendation for an occupational exposure standard for benzene, 1977. 1s Occupational Health and Safety Administration, US. Dept. of Labor, Occupational exposure to benzene, Federal Register 42 (1977)27452-27461. 14 Schmid. W., Chemical mutagen testing on in vivo somatic mammalian cells, Agents and Actions, 3 (1973)77-85. 1 5 Schmid. W.. The micronucleus test for cytogenetic analysis, chapter 36 in: A. Hollaender (Ed.), Chemical Mutagens - Principles and Methods f o r Their Detection. Vol. 4, Plenum, New York. 1976. pp. 31-53. it; .nedecor. G.W., and W.G. Cochran, Statistical Methods, 6th edn.. Iowa State University Press. Ames, ~ w a .1967.p. 272. 1: ,nyder. R., E.W. Lee, J.J. Kocsis and C.M. Witmer, Bone marrow depressant and leukemogenic .&ions of benzene, Life Sci., 21 (1977)1709-1722. 18 Trzos, R.J.. G.L. Petzold, M.N. Brunden and J.A. Swenberg, The evaluation of sixteen carcinogens in the rat using the micronucleus test, Mutation Res., 58 (1978)79-86. 19 Vigliani, E.C., and A. Forni. Benzene and leukemia, Environ. Res., 11 (1976)122-127. 20 Wild, D.,Cytogenetic effects in the mouse of 17 chemical mutagens and carcinogens evaluated b y the micronucleus test, Mutation Res., 56 (1978)319-327. 21 Wolman, S.R.. Cytologic and cytogenetic effects of benzene. J. Toxicol. Environ, Health, Suppl. 2 (1977)63-68. tssistance of Dr. N.R. `eck,both of the Merck , PA. In addition, the !mehius for secretarial .ic hydrocarbons. chapter 9: in Sharp and M.L. Brehm (Eds.), chemical substances, National Education, and Welfare, Rock- :ene in our environment: Clini6. 978). part IV,21838-21854. ols. Mutation Res., 47 (1978) ,SUPPl. 2 (1977)69-105.