Document M4806dE7ewDLBQjpx3Zy6Q17

-4cta haemat. 38: 104-111 (1967) Medical Department and Isotope Laboratory of the Biirgerspital Solothurn (Physician-in-chief: Prof. S. MOESCHLIN) Experimental Studies on the Mechanism of Action of Benzene on the Bone Marrow (Radioautographic Studies Using SH-Thymidine)* S. MOESCHLIN and B. SPECK Despite the fact that the use of benzene as a solvent is prohibited in most countries there are still many reports on chronic benzene intoxications (1, 2, 3, 4, 5). The most frequently quoted sign is aplastic anemia, but also there has been a high incidence of leukemia which generally has been of the acute myeloblastic type. The elective toxic effects of benzene on the bone marrow and the persisting inhibition of the proliferation of the marrow cells even after stopping benzene exposure still remain unanswered questions @a)* I n the present study, the mechanism of action of this substance on the bone marrow cells is examined with tritiated thymidine. Methodr Sixty rabbits with a body weight of 1.1 to 3.7 kg and of both sexes were examined. In preliminary studies 28 animals received benzene in oily solutions subcutaneously. This group of animals could not be evaluated statistically since there was a wide variation of resorption and of the resulting toxic effects. Another group of 5 animals was given a single injection of 2.0 ml/kg of pure benzene subcutaneously. After the initial technical difficulties had been solved, a main experiment was started with 27 animals. This group was intoxicated with subcutaneous injections of pure benzene three times weekly. I t should be mentionned at this point, that the difference between the lethal dose and the one inducing a pancytopenia was small. In our animals, the optimal dose of pure benzene proved to be in the order of 0.3 ml/kg/day. With this dose a severe pancytopenia could be induced in all the animals within one to nine weeks, except for those dying from early anaphylactic or infectious complications. As soon as the leukocyte count in the peripheral blood dropped below 3000/mm8,0 . 5 ~ ~ of aH-methyl-thymidine (New England Nuclear, Boston, Mass.) were given intravenously. The specific activity of this pyrimidine nucleoside labelled with tritium was This work has been supported by Swiss National Grant number 4003. MOESCHLIN/SPECK 105 6.7 C/mM throughout. After an incubation period of exactly 60 min, the animals were anestetized. Bone marrow was aspirated from the epiphysis of the femur. Thin slides were then prepared from solid marrow particles. After air drying, they were fixed in absolute ethanol for 10 min and then covered with Kodak AR-10 stripping film-in the darkroom. The preparations were then exposed for 20 days at 4C. Subsequently they were developped with Kodak D-19 developper and Kodak finator at 18C. The staining was performed through the film with a Giemsa solution buffered to a pH of 6.5. A total number of 19 animals was studied radioautographically. In all these animals 500 basophilic and 500 polychromatophilic normoblasts were enumerated and the percentage of labelled cells was calculated. The pronormoblasts [= K, stage according to WEICKER (7)] and the macronormoblasts ( = K,) could be definitely quantitated only in three intoxicated animals, and the myelocytes only in 2 animals. Cells were considered to be labelled if the number of grains over their nucleus was more than three times the background gaincount over an equal area. Results Peripheral blood. With a dose of 0.3 ml/kg/day of pure benzene, the time span until a peripheral pancytopenia was induced varied from 1 to 9 weeks. A single injection of 2.0 ml/kg did not cause any remarkable changes of the peripheral blood counts during a 6 weeks control period. The differential counts of the leukocytes showed a slight drop of the lymphocytes and a slight increase of basophils and eosinophils. If' the relative percents of the different cell lines were cal- i% 106 MOESCKLIN/SPECEKx,perimental Studies on the Mechanism Hb ing % EC in Mio/mm3 Retl in 9bo -11 - I I4 10 5 10 9- 8- 7- 6- 5- c- 3- 2- 1- aa a = before benzene b = after benzene b ab ab 'a b Fig.2. Mean peripheral blood counts of 19 animals of the main experiment which came to radioautography. The bars at the left represent the values before benzene was injected, the ones on the right the values at the time radioautography was performed. Note again the striking drop of the leukocyte, thrombocyte and the reticulocyte counts with an only slight drop of the erythrocytes and the hemoglobin levels. -100 0 3 - 90. 90. 80- 80- 70. 70- -60. 60 -50 50- -60- LO 30- 20- 20- 20- - --10 10- a)Nmnal control aninals b)Aniials after benzene intox. a)Nmnal camd animals b)pniils after benzene intox Fig.3. Labelling of the young erythroid precurors. Note that there is only a drop of 95.5% to 94.5% labelling a t the pronormoblast ( = K,) stage and of 9 4 O b to 90% a t the macronormoblast ( = K,) stage in normal animals compared to animals intoxicated with benzene. culated in absolute numbers of cells per mms, a decrease of all elements was found, which was most striking in the lymphocytes. Bone marrow. Despite the fact that all the animals had a peripheral pancytopenia after exposure to benzene, there was a considerable difference in the morphologic marrow picture. Of the examined 19 marrows 4 were very hypoplastic, 6 hypoplastic, 5 of average of Action on the Bone Marrow Basophilic 1100.I 90 - 80. 70 . 60. 50. &O - 30 ~ M- 10 - ~ XI. 0- amal a contrd animals b limals Yef rnzene mx Pdychromatcphilic 5 L 3 2 1 bm?dl b contrd animals 107 MOESCHLIN/SPECEKx,perimental Studies on the Mechanism I t . Fig. 5 (a) Radioautographic plate from the marrow of a normal animal. Note the distinct labelling of the basophilic normoblasts. (b) Radioautographic plate from an intoxicated animal made under exactly the same rnnditions. Note the lack of labelling of most of the erythroid precursors. Below, there is a late myelocyte which is not labelled either. The background grain count is low in both preparations. toxicated animals, which persisted at the stage of the polychromatophilic normoblast. The myeloid line was so markedly reduced after exposure to benzene, that only in two animals 500 cells could be quantitated. At stage of the myelocyte the labelling was decreased from 67% in normal animals to 7.3% (s.D. 2.1%) in the intoxicated ones. Discussion .. - 3 : Our radioautographic studies prove that the myelotoxicitr of ,:r i4 ;. benzene and the resulting panvtopenia are caused by a very pronounced t4Q c inhibition of DNA-synthesis. The 3H-thymidine which was used for C:n I r. in vivo incubation is a direct precursor of DNA. During an incuba- I:: :I , tion period of 60 min, all the cells which are in the process of active DNA-synthesis are labelled (8-10).Therefore the amount of labelling of the diyerent cell @pes is a direct index for their proliferative potential. In the erythroid line, the proliferation of the pronormoblasts of Action on the Bone Marrow K2 Pronormoblast --+ Megaloblast K1 hlacronorrnoblart 109 %F Y ir, cc c. -:i* t I t t I i , Polichromatophilic normoblast $$:' ,Oxiphilic normoblast K V 8 Loss o nuc eut Reticulocyte Erythrocyte Fig. 6. Model of erythropoesis (WEICKER)T. he maturation arrest in erythropoiesis during chronic benzene intoxication must be between the stages K, and Ky,. (= K,) and of the macronormoblasts (= K,) was in the normal range as far as this could be quantitated. O n t h e other hand there was a very pronounced lack of proliferative potential at the stage of the basophilic normoblast (= K s ) in all the animals. This finding indicates a maturation arrest between the macronormoblast (= K,) and the basophilic normoblast (= K s ) . Similar observations have been made in other intoxications, e.g. lead (6b). In the myeloid line also, there was a marked decrease of the proliferative potential during benzene intoxication. It was quantitated at the stage of the myelocyte. The short survival time of the leukocytes and of the thrombocytes in the peripheral blood and the rapid maturation of the reticulocytes explain the early drop of these blood counts following severe inhibition of DNA-synthesis. On the other hand the erythrocyte counts and the hemoglobin levels remained relatively constant during benzene intoxication. This is due to the longer survival of the red cells in the peripheral blood. The fact that the human bone marrow may become aplastic as well as hyperplastic during chronic benzene poisoning, is well known (6a, 11). i i I i I i i with antimetabolites (12, 13), aplastic crises of hemolytic anemias and in pernicious anemia (14). Addendum. Since this work has been completed, additional radioautographic studies have been performed in our laboratory, using `H-cytidine and `H-uridine. Our preliminary data indicate that in addition to the inhibition of DNA-synthesis, then may be an inhibition of RNA-synthesis of the bone marrow cells during chronic benzene intoxication. Summary q+., " `-' In rabbits intoxicated by means of subcutaneous benzene injections, numeric studies of the labelled bone marrow cells after intravenous incubation with SH-thymidine were performed. There was a marked individual difference of sensitivity to the same do& of benzene. A severe peripheral pancytopenia resulted in all the animals within one to nine weeks. The bone marrow became hypoplastic in about one half of the animals and it remained cellular in the other half. This is in agreement with the findings in chronic human benzene intoxication. For the first time it was shown radioautographically with `H-thymidine that the pancytopenia of chronic benzene poisoning is due to a severe inhibition of DNA-synthesis in the bone marrow cells. The severe disturbance of DNAsynthesis may also be one of the factors which could give rise to the development of leukemia. &ramrnenfassung Bei Kaninchen mit experimenteller Benzolvergiftung wurde das Knochenmark autoradiographisch untersucht. Die individuelle Empfindlichkeit auf eine gleiche Benzoldosis schwankt erhcblich. Bei dreimaligen s. c. Benzolinjektionen pro Woche konmt es nach 1 bis 9 Wochen zu einer schweren Panzytopenie. Das Knochenmark wurde bei der Halfte der Tiere hypoplastisch, bei den iibrigen war es noch zellreich. Dieses Ergebnis stimmt mit den Beobachtungen bei der menschlichen Benzolvergiftung iiberein. Erstmals konnte durch autoradiographische Untersuchung mit a-HThymidincine schwere Hemmung der DNS-Synthese als Ursache fur die gehemmte Blutzellbildung a bei der Benzolvergiftung nachgewiesen werden. Die schwerwiegende Storung der DNS- .1 Synthese konnte ein Faktor sein, der die leukamixhe Entartung des Knochenmarkcs :.S bei der chronischen Benzolvergiftung begiinstigt. if f Rhurni 5, La moelle osseuse de lapins intoxiqub par des injections souscutankes de benzol a 3 kt6 Ctudite par autoradiographie a p r b l'injection intraveineuse de thymidine tritite. La sensibilitt individuelle P la mtme dose de benzol varie considtrablement. Une pancy- toptnie ptriphtrique stvtre apparut chez tous les animaux au bout de une P neuf se- b r maines, les injections ttant administrkes A raison de trois par semaine. La moelle osseuse devint hypopiasique chez A peu p r b la moitit des animaux; chez les autres, elle demeura riche en cellules. Ces rbultats concordent bien avec les constatations faites dans l a cas d'intoxication au benzol chez I'homme. Pour la premiire fois, il a t t t dkmontrt par auto- radiographie P I'aide de thymidine tritite que la pancytophie de l'intoxication chronique a u benzol est due P une inhibition stvtre de la synthbe de I'ADN. Cette inhibition a t peut-&re aussi I'un des facteurs pouvant favoriser l'apparition d'une IeucCmie lors d'une intoxication chronique au benzol. of Action of Benzene on the Bone Marrow 111 References 1. SCH~NENBEREC.EMR.,:Erneute Benzolvergiftungenin der schweizerischen Uhrenindustrie. Schweiz. med. Wschr. 93: 1469 (1963). 2. GALLINELLI, R.e TRALDIA, . :L'emopatia benzenica. Tre casi di benzolism0 cronico di cui due mortali (leucemia acuta, panmieloftisi acuta).Med. LavoroY: 169 (1963). 3. VIGLIANFI., C. and SAZTAG,.: Benzene and leukemia. New Engl. J. Med. 271: 872 (196$)" 4. TAREEPP, E. M. ;KONTCHALOVSUNY.AM, . and ZORINAL,. A. :Benzene leukemias. Acta Un. int. Cancer 19: 751 (1963). 5. COSCIAG,. C.; F'ERRELLGI., and MEO, G.: Aspetti clinici dell'emopatia da benzolo. Folia med. Napoli 46: 791 (1963). 6. MOESCHLISN.,:Poisoning, Diagnosis and Treatment, 45, 329 (Grune & Stratton, New York, N. Y. 1965). a ) p. 329ff., b) p. 45ff. 7. WEICKERH, .: 5. Kongr. Europ. Ges. Haemat. (Springer, Berlin 1955). 8. FLIEDNERT,. M.; CRONKITE, E. P. und BOND, V. P.: Die Proliferationsdynamik der Blutzellbildung, autoradiographisch untersucht mit tritiummarkiertem Thymidin. Schweiz. med. Wschr. 89: 1061 (1959). 9. COTTIERH, .; ODARTCHENKO, N.; FEINENDECEN, L. E.; KEISERG, . und BOND, V. P.: Autoradiographische Untersuchungen iiber die Entkernung der Erythroblasten nach in vim Markierung mit Thymidin-'H. Schweiz. med. Wschr. 93: 1061 (1963). 10. OEHLERT, W. :Durchfiihrung und Anwendungsmijglichkeitender autoradiogaphixhen Methode in der Pathologie. Schweiz. med. Wschr. 94: 1009 (1964). I 1. MALLORY, T. B. ; GALL, E. A. and BRICKLEYW, . J.: Chronic exposure to benzene. J. Indust. Hyg. and Tox. 21: 355 (1939). 12. SPECK, B.: Morphologic changes in folic acid deficiency induced by methotrexate in human bone marrow and blood. Thesis U.of Minn. Grad. School, 1965. 13. THIERSCHJ,. B.: Bone marrow changes in man after treatment with aminopterin, amethopterin and aminoanfol. Cancer 2: 877 (1948). 14. DOWNEY,H.: The megaloblast-normoblast problem. A cytologic study. J. lab. clin. Med. 39: 837 (1952). Aufhors' oddrcrr: Prof. Sven Moerblin and Dr. Bruno Speck, Medizinirhe Abteilung, BGrgenpital, 4500 Solothurn (Switzerland).