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Mitotic abnormalities in proliferating hepatocytes induced by thioacetamide at certain periods after partial hepatectomy
TAA-induced REFERENCES Lillie, R D, Histopathologic technic and practical histochemistry, 3rd edn. McGraw-Hill, New York (1965).
MITOTIC ABNORMALITIES IN PROLIFERATING HEPATOCYTES INDUCED BY THIOACETAMIDE AT CERTAIN PERIODS AFTER PARTIAL HEPATECTOMY ST. MIRONESCU, Department of Morphology, Institute sf Endocrinology, Bucharest, Roumania Thioacetamide (TAA) elicits in rat liver striking changes in both interphasic and dividing hepatocytes. The nucleolar [IO-12, 161 and mitotic [14] alterations are two of the most prominent, and some of them were linked to the carcinogenic action of this drug [14, 161. Repeated applications are necessary to induce these morphological changes, since, as indicated by the studies in which 35S- and 3H-labeled TAA was used, during the first 24 h this drug is rapidly converted to acetate, via the acetamide pathway [20], and excreted in the urine [19,20]. Due to the rapid metabolism of TAA, the maximum and most uniform cytological effects were found 4 h after its subcutaneous administration [lo, 121. The relative rapid degradation of TAA within the liver tissue [20] and the possibility to obtain, after the subtotal liver resection, a sequence of macromolecular syntheses, mimming during the first 31 h, the ordered biochemical events occurring in the cell cycle [l-4], offers exploitable circumstances to test (a) whether single doses of TAA may induce changes in the regenerating liver, and (6) whether the biosynthetic steps, leading to a wave of synchronous DNA replication and mitoses [l, 41, exhibit variable degrees of sensitiveness toward the damaging action of this compound. The limits of interpretation of the biochemical changes developed in regenerating rat liver, concerning their resemblance to the processes operating during the cell life cycle of unstimulated hepatocytes, were outlined earlier [IS] and, more recently, they were extensively reviewed [2].
Materials
abnormalities
in hepatocytes
435
and methods
Partially hepatectomized [9] male Sprague-Dawley rats (89k3.4 g) divided in four groups of 8, 8, 6 and 12 animals, were inoculated intra-abdominally at 10, 20, 23 and respectively 26 h after operation with appropriate volumes of a 1 % solutionof TAA in 0.14 M NaCl, to achievein therecipientsthedrugconcentrationof 150mg/ g body weight.The two control rats of eachgroupreceivedappropriatevolumes of physiologicsaline.All the control and experimentalanimalswerekilled 31 h after surgery,between9-10a.m.,andthe remnantliver tissues werefixed in 10%neutralbufferedformaldehyde. Twenty p thick paraffin sections were prepared and stained by the Feulgenprocedure.At leastthree sectionsof each animal were checked for the presence of mitotic abnormalities (single or multiple chromosome bridges, acentric chromosome fragments and lagging chromosomes) in the anaphasic and telophasic stages of mitosis. At least 200 anaohases and telouhases were studied for each tested rat and’ the results were expressed as the mean number of abnormal anaphases and telophases/lOO anaphases and telophases. Thk data obtained-from the control rats were pooled and treated as for a single specimen. Statistical analyses were performed using Student’s t-test. The significance threshold was chosen at the level of P < 0.05.
Results Fig. 1 presents the variations of the different morphological types of abnormal anaphasesand telophases, occurring in regenerating rat liver cells after a single injection of 150 mg TAAjkg b.w. The data recorded in the controls showed that in saline-treated Sprague-Dawley rats, the occurrence of abnormal anaphases and telophases ranged between 7.5-8.5% of the total number of anaphasesand telophasesencountered 31 h after partial hepatectomy. Among these abnormalities 40-60 % were represented by the chromosomal bridges, about 30 % by the lagging chromosomes and lo-30 % by the acentric chromosome fragments. Compared with the control animals, the rats receiving 150 mg TAA/kg b.w., 10 h after they were partially hepatectomized, presented significantly increased proportions of both chromosome bridges and acentric chromosome fragments. Although increased, the frequency of the lagging chromosomes was found not statistically different from the values recorded in control rats, and this was also valid for all other intervals studied. The rats inoculated with TAA 20 h after surgery have presented extremely high proportions of chromosomal bridges, 50-75 % of anaphasic Exptl
Cell Res 5.5
436
G. E. Levenson
I955 IO
11.56
II29
20
23
31
31
31
Fig. 1. Changes in the frequency of thedifferent morphological types of abnormal anaphases and telonhases in regenerating livers of rats receiving a single injkction of TAA (150 mg/kg b.w.) at various times after partial hepatectomy. The statistically assured differences against the control values are indicated upper the respective columns. Abscissa: Hours after parial hepatectomy; ordinate: no. of abnormal anaphases and telophases/total no. of anaphases and telophases x 100.0, bridges; m, acentrics; E?J, loggards; 1, range.
and telophasic figures displaying this mitotic abnormality. Contrasting with the other intervals tested, the bridges recorded at this time were thicker and often of multiple type. The frequency of anaphasic and telophasic chromosome bridgesfall consistently in regenerating livers of rats receiving TAA 23 h after operation. However,
their
percentual
values
remained
significantly above the control level. When TAA was given 26 h after partial hepatectomy the yield of abnormal anaphases and telophases was not different from that recorded in control saline-treated rats. Discussion From the data reported it appears that during the first 31 h after partial hepatectomy, the regenerating hepatocytes pass through phases of different sensitivenesstoward the TAA ability to induce mitotic abnormalities. Both autoradiographic [7] and biochemical Expti Cell Res 55
[3, 41 studies demonstrated that in rats, at 20 h after 213 removal of the liver mass, the remnant parenchymal cells are engagedin DNA synthesis. Quantitative estimations pointed out that in the first 24 h of the postoperative period about 5560 % of the hepatocytes are stimulated to enter DNA synthesis [5]. This is achieved gradually, beginning from the 13 h after operation, and the maximum rate of DNA synthesis as well as the greatest number of cells exhibiting this process were encountered 21 and, respectively, 23 h after partial hepatectomy [ 121. Thus, it seemsvery likely that TAA inoculated 20 h after surgery reached the parenchymal liver cells, found in mitosis at 31 h, while they were in the DNA replicative period. On these grounds it may be supposedthat the hepatocytes during the phase of stimulated DNA synthesis are highly sensitive to the mitotic damaging effects of TAA. The drastically reduced yield of anomalous anaphasesand telophaseswhen TAA was inoculated 23 and 26 h after partial hepatectomy tend to demonstrate that the stagesthrough which the proliferating hepatocytes, after completing their DNA manufacture, pass toward the subsequent division, were more resistant to the genomedamage induced by this drug. It seemsto be remote that the reduced time elapsed between TAA inoculation and the sacrifice would influence the magnitude of the response. In both these cases this time-length was longer than 4 h and moreover, the significant difference against the controls of rats inoculated at 23 h with TAA, as well asthe great variation of the values recorded when TAA was administered 26 h after operation, proved that this compound was also still active in these instances. The significant differences versus the controls, recorded in the rats inoculated 10 h after partial hepatectomy permitted us to assumethat the prereplicative periods of stimulated hepatocytes may be also sensitive, but to a lesserdegree than the DNA synthetic period, toward the abnormal mitosis-inducing properties of TAA. Mechanism(s) similar to that described here could be tentatively forwarded as a possible explanation of the reported findings [6, 7, 16, 17,
TAA-induced abnormalities 211, indicating that a series of carcinogenic drugs act more effectively in inducing malignant growth if they are in contact with cells synthesizing DNA. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Baserga, R, Cancer res 25 (1965) 581. - Cell tissue kinetics 1 (1968) 167. Beltz, R E, Van Lanker, J & Potter, V R, Cancer res 17 (1957) 688. Bucher, N L R, New Engl j med 277 (1967) 686. Fabrikant, J I, J cell biol 36 (1968) 551. Frei, J V & Harsono, T, Cancer res 27 (1967) 1482. Frei, J V & Ritchie, A C, J natl cancer inst 32 (1964) 1213. Grisham, J W, Cancer res 22 (1962) 842. Higgins, G M & Anderson, R M, Arch path01 12 (1931) 186. Kleinfeld, R G, Cancer res 17 (1957) 954.
in hepatocytes
437
11. Kleinfeld, R G & Von Haam, E, Cancer res 19 (1959) 769.
12. Koulish, S & Kleinfeld, R G, J cell biol23 (1964) 39. 13. Looney; W B, Chang,.L 0 & Banghart, F W, Proc natl acad sci US 57 (1967) 972. 14. Maini, M M & Stich, H F, J natl cancer inst 26 (1961) 1413. 15. Mironescu, St & Dragomir, C, Cancer res 27 (1967) 1819. 16. Mironescu. St. Encut. I. Mironescu. K & Liciu. I F.I ’ J natl cancer inst 40 (1968) 917. 17. Mottram. J C. J path01 bacterial 56 (1944) 391. 18. -Ibid 57 (1945)265. 19. Nygaard, 0, Eldjarn, L & Nakken, K F, Cancer res 14 (1954) 625. 20. Rees, K R, Rowland, G F & Varcoe, J S, Intern j cancer 1 (1966) 197. 21. Shinozuka, H & Ritchie, A C, Intern j cancer 2 (1967) 77.
Received February 3, 1969
Exptl
Cell Res 55
MITOTIC ABNORMALITIES IN PROLIFERATING HEPATOCYTES INDUCED BY THIOACETAMIDE AT CERTAIN PERIODS AFTER PARTIAL HEPATECTOMY ST. MIRONESCU, Department of Morphology, Institute sf Endocrinology, Bucharest, Roumania Thioacetamide (TAA) elicits in rat liver striking changes in both interphasic and dividing hepatocytes. The nucleolar [IO-12, 161 and mitotic [14] alterations are two of the most prominent, and some of them were linked to the carcinogenic action of this drug [14, 161. Repeated applications are necessary to induce these morphological changes, since, as indicated by the studies in which 35S- and 3H-labeled TAA was used, during the first 24 h this drug is rapidly converted to acetate, via the acetamide pathway [20], and excreted in the urine [19,20]. Due to the rapid metabolism of TAA, the maximum and most uniform cytological effects were found 4 h after its subcutaneous administration [lo, 121. The relative rapid degradation of TAA within the liver tissue [20] and the possibility to obtain, after the subtotal liver resection, a sequence of macromolecular syntheses, mimming during the first 31 h, the ordered biochemical events occurring in the cell cycle [l-4], offers exploitable circumstances to test (a) whether single doses of TAA may induce changes in the regenerating liver, and (6) whether the biosynthetic steps, leading to a wave of synchronous DNA replication and mitoses [l, 41, exhibit variable degrees of sensitiveness toward the damaging action of this compound. The limits of interpretation of the biochemical changes developed in regenerating rat liver, concerning their resemblance to the processes operating during the cell life cycle of unstimulated hepatocytes, were outlined earlier [IS] and, more recently, they were extensively reviewed [2].
Materials
abnormalities
in hepatocytes
435
and methods
Partially hepatectomized [9] male Sprague-Dawley rats (89k3.4 g) divided in four groups of 8, 8, 6 and 12 animals, were inoculated intra-abdominally at 10, 20, 23 and respectively 26 h after operation with appropriate volumes of a 1 % solutionof TAA in 0.14 M NaCl, to achievein therecipientsthedrugconcentrationof 150mg/ g body weight.The two control rats of eachgroupreceivedappropriatevolumes of physiologicsaline.All the control and experimentalanimalswerekilled 31 h after surgery,between9-10a.m.,andthe remnantliver tissues werefixed in 10%neutralbufferedformaldehyde. Twenty p thick paraffin sections were prepared and stained by the Feulgenprocedure.At leastthree sectionsof each animal were checked for the presence of mitotic abnormalities (single or multiple chromosome bridges, acentric chromosome fragments and lagging chromosomes) in the anaphasic and telophasic stages of mitosis. At least 200 anaohases and telouhases were studied for each tested rat and’ the results were expressed as the mean number of abnormal anaphases and telophases/lOO anaphases and telophases. Thk data obtained-from the control rats were pooled and treated as for a single specimen. Statistical analyses were performed using Student’s t-test. The significance threshold was chosen at the level of P < 0.05.
Results Fig. 1 presents the variations of the different morphological types of abnormal anaphasesand telophases, occurring in regenerating rat liver cells after a single injection of 150 mg TAAjkg b.w. The data recorded in the controls showed that in saline-treated Sprague-Dawley rats, the occurrence of abnormal anaphases and telophases ranged between 7.5-8.5% of the total number of anaphasesand telophasesencountered 31 h after partial hepatectomy. Among these abnormalities 40-60 % were represented by the chromosomal bridges, about 30 % by the lagging chromosomes and lo-30 % by the acentric chromosome fragments. Compared with the control animals, the rats receiving 150 mg TAA/kg b.w., 10 h after they were partially hepatectomized, presented significantly increased proportions of both chromosome bridges and acentric chromosome fragments. Although increased, the frequency of the lagging chromosomes was found not statistically different from the values recorded in control rats, and this was also valid for all other intervals studied. The rats inoculated with TAA 20 h after surgery have presented extremely high proportions of chromosomal bridges, 50-75 % of anaphasic Exptl
Cell Res 5.5
436
G. E. Levenson
I955 IO
11.56
II29
20
23
31
31
31
Fig. 1. Changes in the frequency of thedifferent morphological types of abnormal anaphases and telonhases in regenerating livers of rats receiving a single injkction of TAA (150 mg/kg b.w.) at various times after partial hepatectomy. The statistically assured differences against the control values are indicated upper the respective columns. Abscissa: Hours after parial hepatectomy; ordinate: no. of abnormal anaphases and telophases/total no. of anaphases and telophases x 100.0, bridges; m, acentrics; E?J, loggards; 1, range.
and telophasic figures displaying this mitotic abnormality. Contrasting with the other intervals tested, the bridges recorded at this time were thicker and often of multiple type. The frequency of anaphasic and telophasic chromosome bridgesfall consistently in regenerating livers of rats receiving TAA 23 h after operation. However,
their
percentual
values
remained
significantly above the control level. When TAA was given 26 h after partial hepatectomy the yield of abnormal anaphases and telophases was not different from that recorded in control saline-treated rats. Discussion From the data reported it appears that during the first 31 h after partial hepatectomy, the regenerating hepatocytes pass through phases of different sensitivenesstoward the TAA ability to induce mitotic abnormalities. Both autoradiographic [7] and biochemical Expti Cell Res 55
[3, 41 studies demonstrated that in rats, at 20 h after 213 removal of the liver mass, the remnant parenchymal cells are engagedin DNA synthesis. Quantitative estimations pointed out that in the first 24 h of the postoperative period about 5560 % of the hepatocytes are stimulated to enter DNA synthesis [5]. This is achieved gradually, beginning from the 13 h after operation, and the maximum rate of DNA synthesis as well as the greatest number of cells exhibiting this process were encountered 21 and, respectively, 23 h after partial hepatectomy [ 121. Thus, it seemsvery likely that TAA inoculated 20 h after surgery reached the parenchymal liver cells, found in mitosis at 31 h, while they were in the DNA replicative period. On these grounds it may be supposedthat the hepatocytes during the phase of stimulated DNA synthesis are highly sensitive to the mitotic damaging effects of TAA. The drastically reduced yield of anomalous anaphasesand telophaseswhen TAA was inoculated 23 and 26 h after partial hepatectomy tend to demonstrate that the stagesthrough which the proliferating hepatocytes, after completing their DNA manufacture, pass toward the subsequent division, were more resistant to the genomedamage induced by this drug. It seemsto be remote that the reduced time elapsed between TAA inoculation and the sacrifice would influence the magnitude of the response. In both these cases this time-length was longer than 4 h and moreover, the significant difference against the controls of rats inoculated at 23 h with TAA, as well asthe great variation of the values recorded when TAA was administered 26 h after operation, proved that this compound was also still active in these instances. The significant differences versus the controls, recorded in the rats inoculated 10 h after partial hepatectomy permitted us to assumethat the prereplicative periods of stimulated hepatocytes may be also sensitive, but to a lesserdegree than the DNA synthetic period, toward the abnormal mitosis-inducing properties of TAA. Mechanism(s) similar to that described here could be tentatively forwarded as a possible explanation of the reported findings [6, 7, 16, 17,
TAA-induced abnormalities 211, indicating that a series of carcinogenic drugs act more effectively in inducing malignant growth if they are in contact with cells synthesizing DNA. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Baserga, R, Cancer res 25 (1965) 581. - Cell tissue kinetics 1 (1968) 167. Beltz, R E, Van Lanker, J & Potter, V R, Cancer res 17 (1957) 688. Bucher, N L R, New Engl j med 277 (1967) 686. Fabrikant, J I, J cell biol 36 (1968) 551. Frei, J V & Harsono, T, Cancer res 27 (1967) 1482. Frei, J V & Ritchie, A C, J natl cancer inst 32 (1964) 1213. Grisham, J W, Cancer res 22 (1962) 842. Higgins, G M & Anderson, R M, Arch path01 12 (1931) 186. Kleinfeld, R G, Cancer res 17 (1957) 954.
in hepatocytes
437
11. Kleinfeld, R G & Von Haam, E, Cancer res 19 (1959) 769.
12. Koulish, S & Kleinfeld, R G, J cell biol23 (1964) 39. 13. Looney; W B, Chang,.L 0 & Banghart, F W, Proc natl acad sci US 57 (1967) 972. 14. Maini, M M & Stich, H F, J natl cancer inst 26 (1961) 1413. 15. Mironescu, St & Dragomir, C, Cancer res 27 (1967) 1819. 16. Mironescu. St. Encut. I. Mironescu. K & Liciu. I F.I ’ J natl cancer inst 40 (1968) 917. 17. Mottram. J C. J path01 bacterial 56 (1944) 391. 18. -Ibid 57 (1945)265. 19. Nygaard, 0, Eldjarn, L & Nakken, K F, Cancer res 14 (1954) 625. 20. Rees, K R, Rowland, G F & Varcoe, J S, Intern j cancer 1 (1966) 197. 21. Shinozuka, H & Ritchie, A C, Intern j cancer 2 (1967) 77.
Received February 3, 1969
Exptl
Cell Res 55