Suppression of Cellular Activity in the Reticuloendothelial System of the Rat by 7,12-Dimethylbenz(A)Anthracene 1

[CANCER RESEARCH 28, 320-327, February 1968] Suppression of Cellular Activity in the Reticuloendothelial System of the Rat by 7,12-Dimethylbenz(a)anthracene 1

Madison J. Cawein ~ and Katherine L. Sydnor With the technical assistance of Dona Dunne, Thomas Roach, and Yvonne Spencer Department oJ Medicine, University of Kentucky College oJ Medicine, Lexington, Kentucky ~0506

SUMMARY MATERIALS AND METHODS

A single feeding of 7,12-dimethylbenz (a) anthracene (DMBA) Female Spraguc-Dawley rats, age 50 days, were used in five (112 or 133 mg/kg) to female Sprague-Dawley rats, age 50 separate experiments. days, induced maturation arrest at the proerythroblast, pro- Blood for hematologic indices was obtained from the tail myeloblast, and promegakaryocytoblast level in bone marrow, vein. Microhematocrits were performed in duplicate; leukocyte depletion of thymic lymphocytes, and disorganization of the and reticulocyte counts were determined by standard methods; lymphoid follicles in the spleen. The peripheral blood reflected one hundred leukocytes were counted for differential counts. these changes. A decrease in lymphocytes was first observed Platelets were counted by the phase contrast method of Brecher on Day 2 after DMBA. At this time the total granuloeytc and Cronkite (6). Erythrocyte ghlcose-6-phosphate dehydro- count was increased. Lymphocytopenia and granulocytopenia genase (G-6-PD) was assayed by the method of Marks (22), were maximal on Days 5 and 6, but granulocytopenia was more erythrocyte reduced glutathione (GSH) by the method of profound. Thrombocytopenia and its attendant hemorrhagic Beutler e~ al. (5), and serum acid phosphatase (SAP) by the phenomena were maximal between Days 9 and 15. Morpho- method of Andersch and Szczypinski (2). Methemoglobin was logic changes indicative of damage were first apparent in mar- determined by the method described by Evelyn and Malloy row on Day 3 and of recovery from damage on Day 9; regen- (14). DMBA-ZH was prepared by Dr. James Flesher, Depart- erative changes occurred rapidly after Day 15 and were ment of Pharmacology; radioassay of tissues was conducted by virtually complete by Day 25. There was no evidence for direct the method described by Hamilton and Jacobsen (16). Meas- damage to the formed elements in peripheral blood by this urement of 59Fe uptake by reticulocytes in vitro was made in a hydrocarbon. These changes closely mimic those induced by well type scintillation counter with an efficiency for Fe of 10% radiation and are probably the consequence of damage to DNA against appropriate standards. of the proerythroblast, myeloblast, and megakaryoblast precursors. RESULTS Experiment I. INTRODUCTION Influence of DMBA on Organ and Body Weight, Blood Indices and Enzymes. In the first experiment., all experimental Damage to the hematopoietic and lymphoid systems in ex- animals were given a single feeding of DMBA, 133 mg/kg, in perimental animals following treatment with 7,12-dimethyl- sesame oil by gastric tube. Control rats received sesame oil benz(a)anthracene (DMBA) has been reported by several in- alone by the same route. One-half of each group, experimental vestigators (18, 21, 24, 25), but the sequence of events which and control, was allowed to drink tap water; the other half occur in the hematopoietic system have not been described suf- drank 0.24% oxytetracycline. Two groups of rats, experimental ficiently to show a specific pattern of response. It is the purpose and their appropriate controls, were randomly selected for of this communication to report the sequential changes which sacrifice 1, 2, 3, 6, 9, 12, 15, 20, and 25 days after DMBA occur in this system in the female Sprague-Dawley rat given a feeding. Blood for microhematocrit, and leukocyte and platelet single feeding of DMBA. Our observations indicate that this determinations was obtained from the tail vein immediately hydrocarbon induces a profound but reversible maturation prior to sacrifice. Blood for enzyme determinations was col- arrest of erythropoietic and granulocyte precursors under the lected under ether anesthesia from the abdominal aorta via a experimental conditions herein described. polyethylene catheter at time of sacrifice. Sufficient marrow for fresh smears and fixed sections was obtained from the x This investigation was supported by USPHS Research Grant femora. Spleen, thymus, adrenal glands, liver, ovaries, and No-CA-07321. adenohypophysis from each animal were weighed, and selected 2 Present address: Medical Department, The Norwich Pharmacal tissues were fixed in 10% neutral formalin and prepared for Company, P.O. Box 191, Norwich, New York. microscopic examination. An additional group of rats was Received January 11, 1967; accepted October 15, 1967. given 1 mg hydrocortisone acetate by the intramuscular route

320 CANCER RESEARCH VOL. 28

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1968 American Association for Cancer Research. DMBA Suppression el Cellular Activity in the Rat every 12 hours for four doses, then every 24 hours for two rocyte T~ is due to loss of blood into tissues. The total leu- doses. DMBA was fed 20 hours after the initial dose of hydro- kocyte count was significantly reduced 48 hours after DMBA. cortisone. These animals were bled from the tail vein for blood The full magnitude of the changes on Days 1, 2, and 3 are indices on I)ay 9 and sacrificed on Day 25 after DMBA. masked to some extent by hemoconcentration. Maximal leu- Mortality due to the toxic effects of this hydrocarbon was kopenia had occurred by Day 6 and did not change appre- 29% and death occurred during the first week. Early mani- ciably until after Day 15. By Day 20 the total count was higher festations of toxicity--marked hyperemia of the skin and gut, but rem,fined significantly lower than that of the control groups loss of appetite and body weight., and hemorrhagic adreno- as late as Day 25, observations in agreement with those re- cortical necrosis--were first apparent on Day 2. Diarrhea ported by Huggins eta[. (18). The leukocyte T.~ was estimated occurred on Day 3. By Day 4 the overt signs of acute toxicity to be 4.6 days. had disappeared, and the animals appeared vigorous and Peripheral Blood Smears. By Day 3 there was an overall healthy. Since observations on the two groups of animals were reduction in leukocytes, but the relative percentage did not in agreement, they were combined -rod are presented in Tables change. The most remarkable change was observed in the 1 (organ and body weights) and 2 (hematologic indices and polymorphonuelear neutrophils, which were huge with hyper- enzyme activities). segmented nuclei. Normal azurophilic granulation had disap- Peripheral Blood Counts. The mean platelet count of nine peared and the few granules that remained were heavy and groups of control rats ranged from 8.72 to 10.23 X 105 per dark. The cytoplasm was blue. Peripheral blood smears of nor- cu ram. Two days after DMBA the platelet count was signifi- real rats contain two to three polychromatophilie erythrocytes cantly decreased. Bv Day 9 it was 1.72 X 10 .5 and did not per oil immersion field. Normal polychromasia had disappeared, change appreciably until after Day 15. 13y Day 20 thrombocy- and reticuloeytes were absent. By Day 6 virtually all leuko- topenia was less severe and by Day 25 the platelet counts from cyte~ had disappeared and plat.elets were decreased in number. experiment:fl :rod control groups were not significantly dif- By I)'ly 9 some evidence of recovery was apparent. Granu- ferent. Gross evidence of thrombocytopenia was present in all locytes were present, but approximately 50% of the poly- experimental groups sacrificed on Days 9, 12, and 15. Ilemor- morphonuclear leukocytes still showed hypersegmentation. rhagie areas in thymus and lymph nodes were observed in Platelets were few in number, large, and appeared to lack every animal; peteehial hemorrhages in skin and serosal sur- adhesiveness. In most eases polychromasia had reappeared, but faces occurred in animals whose I)latelet counls were 10(),000/ was less than normal. By Day 12 2-4 erythrocytes per oil cu mm or less. Of the ten experimental rats killed on Dav immersion field were polychromatophilic, but. granulocytopenia 15, hemorrhage occurred in the brain of three, in the uterus trod thrombocytopenia persisted. By Day 15, erythroeytes ex- of one, and in the peritoneal cavity of one. In contrast to hibited marked polychromasia with regenerqtive maeroeytosis. adrenocortieal necrosis, which occurs early, the hemorrhagic There were numerous micropherocytes and nucleated eryth- diathesis descrit)ed above occurs late and appears to be the roblasts. Reticulocytes (0.1-45%) were again observed. Leu- consequence of thromboeytopenia. Hydrocortisone had no in- kopenia was still evident. Less than 10% of the cells were fluence on thrombocytopeifia induced by DMBA. An overall neutrophils and many of these cells were hypersegmented. The estimate of platelet T~ is 6 to 7 days and the rate of recovery cytoplasm of the lymphocytes was shaggy and was being shed; was of lhe same order. This estimate is approximate since the the nucM exhibited indentations with occasional bilobing and curve for plateh't disappearance is not strictly linear. Studies a few cells had double nuclei. On Day 20 marked erythroid on nmgakaryoI)oiesis in the rat (10, 15) permit the assumption regener'Kion was observed. Many (10-12 per oil immersion that few platelets would be added to the circulation 72 hours fiehi) regenerative polychromatophilic macroerythrocytes and after production stops; if T~ is calculated on the basis of the nucleMed erythroeytes were observed. The number of poly- 3-, 6-, and 9-day counts, it turns out to be 4.1 days and is morphonuclear leukocytes had increased, but was considerably considerably longer than the time reported for rat platelets less "than th'~t observed in the control group. Platelets were labeled with radioisotopes (8). Our observations may be in- still reduced in number, larger than normal with many giant terpreted to indicate that labeled transfused platelets had a forms, and lacked adhesiveness. By Day 25 the blood smears shorter half-life or that megakaryopoiesis wa.s not totally ar- from apt)roximately one-half the animals were normal. The rested in these animals. The mean hematoerit for nine groups remaining htdf still showed signs of erythroid regeneration of control rats ranged from 46.2 _--_ 0.35 to 48.0 ----- 0.65. On characterized by increase in polyehromasia and many nucleated I)ays 1, 2, and 3 after DMBA, the mean hematocrit was signifi- erythrocytes. No abnormalities were observed in the leukoeytes canth" higher than in the control groups. This is due, at least or platelets. in part, to dehydration. By Day 6 it had decreased to 42.4 ----- Bone Marrow. Smears of marrow reflected the changes ob- 0.8, and by Day 15 to 21.8 __+ 4.18. Recovery thereafter was served in peripheral blood. On Day 3 the number of myelocytie rapid: 36.0 ___ 3.45 on Day 20 and 46.1 __ 0.51 on Day 25. precursors (myeloblasts and promyeloeytes) were decreased The "l'.'~Cr of erythroeytes in Sprague-Dawley rats is ap- and many of the myclocy~es appeared to be arrested. Abnor- proximately 18 days (26). In this experiment, the erythrocyte mally large metamyeloeytes with large ring nuclei exhibiting T~, calculated on the basis of hematocrit, is 15.6 days. Changes multiple areas of pinching (similar to those described in per- in hematocrit after I)MBA represent at least two processes-- nieious anemia by Herbert) and, at times, "figure of eight" maturation arrest of erythroid precursors and hemorrhage due nuclei were observed. The cytoplasm of these cells was im- to thrombocytopenia. We believe the slightly shorter eryth- mature and resembled that of the promyelocytes in that it

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1968 American Association for Cancer Research. M. J. Cawein and K. L. Sydnor stained deep blue and contained large purple granules. A rela- cant increase in serum acid phosphatase activity in the control tive increase in the number of reticulum cells was observed groups sacrificed on Days 9, 12, and 15. and many of them contained phagoeytized material. Mega- Thymus. The weight of the thymus was significantly de- karyoeytes had decreased in number. Many showed failure of creased within 24 hours after DMBA, reached its minimmn platetet formation; a relative increase in bare degenerating ~t, 48 hours, and remained at this low value until after Day 6. megakaryoeytic nuclei was present. By Day 6 severe eryth- On Day 9 the mean weight of thymi from experimental and roeyte hypoplasia had occurred and the remaining erythrocyte control groups were in agreement, but involution occurred a precursors had a megaloblastoid appearance. In a few of the second time in the groups given DMBA and did not return marrows there was an increase in eosinophilic myeloeytes. The to normal until Day 20. Initial decrease in thymus weight may granuloeytie precursors were arrested at the level of the pro- be due, in part, to increased seeretion of adrenoeorticosteroids myeloeyte. Nuclear-cytoplasmic asynchronism was observed, (9) and, in part, to loss of body weight. The second decline i.e., the nucleus was still quite primitive and contained a large cannot be attributed altogether to changes in body weight, nucleolus, while the cytoplasm showed granulation. Mega- for control animals of approximately the same weight have karyocytes were few in number. Some evidence of recovery much heavier thymi. Microscopic examination showed a pro- m the erythroid line w'~s observed on Day 9 but the erythroid found reduction in the number of small lymphocytes. precursors remained depressed. The granuloc, ytic series still Spleen. Mean spleen weight was significantly decreased 24 showed maturation arrest and megaka, ryocytes were few in hours after DMBA and remained so through Day 15. On number. By Day 12 repopulation with erythroid elements had Days 20 and 25 spleens from rats given DMBA were slightly, occurred, but the granulocytie line still showed maturation but not significantly, higher than their controls. Unlike the arrest.. A modest increase m megakaryoblasts was observed on thymus, biphasic weight changes were not observed. No micro, an occasional smear but, in general, megakaryoeytes were scopic changes were observed in spleens removed 24 hours severely depressed. In general, hypoplasia was still severe by after DMBA, but by 48 hours the lymphocytes in the red Day 15. There was a relative increase in retieulum cells, many pulp he~d deereased in number and the lymphoid follicles had with phagocytized nuclei and cellular debris. Many of the decreased in size. Megakaryocytes, abundant on control sec- erythroid precursors had a megaloblastoid appearance. The tions, had ahnost entirely disappeared. This morphologic pat- granutocytic series was arrested and aga.in nuclear-cytoplasmic tern persisted through Day 9. By Day 12 numerous foci of asynchronism was observed. Gross morphological deformity, of lymphocytic cells were observed in the red puIp, but the the developing nuclei of these cells indicated disturbance in prominent follicular pattern characteristic of spleens from con- nuclear maturation. Megakaryocytes were decreased in num- trol rats was absent; megakaryocytes were again observed for ber. Formalin-fixed sections showed a reduction of cellular the first time. Normal spleen morphology was not observed elements and increase in fat, hemosiderin, and fibroblasts, but until Day 20. the fibrotic areas were vascularized. By Day 20 an increase in Body Weight. Changes in mean body, spleen, and thymus over-all cellularity was observed. The erythroid elements were weights are shown in Table 1 for control rats and for the groups given DMBA. Initial weight loss, whieh occurs during the first hyperactive, showing full recovery. A return t.o a more normal three days after DMBA, may be attributed to dehydration maturation sequence had occurred in the granuloey~ie series. and failure to eat.. The second period of weigh~ loss, which Megakaryocytes, though deereased in number, were more occurs between Days 9 and 20, is correlated directly with the abundant. By Day 25, the marrows from experimental animals period of hemorrhagic diathesis. could not be distinguished from those from control groups. It Other Tissues. Changes m adrenal gland morphoIogy after is assumed that complete recovery had occurred at this time. DMBA have been amply described by Huggins and Morii Enzyme Activities. Erythrocyte G-6-PD activity did not (19). Our observations differ in that adrenal calcification had differ from control values until D'ty 20 when it increased to occurred in six of ten rats by Day 9. Liver weight was sig- 27.3 units versus 21.4 for controls. At. no time were the dif- nificantly reduced only on Day 3. In contrast ~o the spleen/ ferences between control aml experimental groups highly ,~io'-,, body weight ratio (rag organ weight/am body weight) which nificant. However, comparison of G-6-PD activity with the was significantly lower than that of control groups, liver/body reticuloeyte count, potyehromasia, and circulating erythro- weight ratio was always higher. The mean weight, of pituitaries blasts from a given animal showed a high degree of correla- from experimental and eont.rol groups was in agreement, de- tion. Changes in erythroeyte GSH activity were not marked, spite a significant reduction in ovarian and uterine weight on but were higher than control values on Days 9, 12, and 15, Days 6 and 9. The latter changes are difficult to interpret at but only on Day 15 were the differences between control (77.2 *he present time. We have observed prolonged va~in,d bleed- _+_ 2.3) and experimental (89.1 __+ 3.4) groups highly significant ing due to uterine hematomata in Sprague-Dawley strain rats (P -- 0.001). These enzyme changes are in general agreement as late as five months after feeding a single 20-rag (lose of with those reported for rats, rabbits, and dogs with secondary, DMBA; Wong eta/. (27) have described ischemic necrosis of anemia and reticulocytosis induced by phenylhydrazine. Serum reeent, ly matured ovaries from rt~ts of this strain similarly acid phosphatase decreased significantly from Day 6 to 15, treated. These changes were observed only on Days 3 and 4 indieating that thrombocytopenia induced by DMBA is a after DMJ3A. The weight changes observed in our experiment consequence of maturation arrest rather than platelet destruc- on Days 6 and 9 are most likely clue to ovarian damage and tion (23). There is no satisfactory explanation for the signifi- its consequences. Ovarian/body weight ratios varied between

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Table 1

Day Initial Final after No. of body wr body wt. Thymus Spleen Adrenals Ovaries Uterus Pituitary Liver DMBA rats (gin) (grn) (rag) (rag) (mg) (mg) (mg) (mg) (gin) 1 Control 7 173 • 3 177 __+ 3 412 __+ 27 512 __+ 25 53 • 7.8 92 4- 4.3 347 • 30 11.8 • 0.9 6.5 __+ 0.2 DMBA 12 177 ___ 3 172 -+- 3 326 -+- 21 ~27 • 25 51 • 4.3 91 ___ 3.0 317 • 23 10.0 ~ 0.5 6.7 ___ 0.2

2 Control 7 171 ___ 2 174 -+- 2 393 ~ 9 478 4- 23 54 ___ 2.5 92 • 3.3 340 ___ 29 12.0 4- 0.6 6.6 +__ 0.3 DMBA 12 183 4- 4 173 4- 3 270 4- 15 ~12 ~ 18 86 4- 5.~ 92 +__ 3.1 297 + 23 12.0 4- 0.4 6.9 4- 0.3 3 Control 10 166 • 4 173 ___ 4 379 ___ 15 595 ___ 27 47 4- 2.2 86 4- 4.6 307 ___ 19 9.3 __+ 0.3 6.8 + 0.3 DMBA 12 173 -+- 3 153 4- 4 256 4- 17 327 4- 19 89 "4- 42 83 • 4.6 2~2 4- 12 9.8 4- 0.4 5.9 4- 0.2

6 Control 10 164 4- 5 183 __. 2 414 ___ 22 563 -+- 19 52 ___ 5.8 100 • 3.4 331 4- 12 10.5 -+- 0.5 6.9 • 0.2 DMBA 12 166 ___ 3 171 4- 3 250 -+- 18 387 -~ 57 102 -4- ~,.9 80 ~ 4.7 2~1 4- 20 9.4 __+ 0.4 7.5 ___ 0.3

9 Control 10 166 -+- 3 192 • 2 373 ___+19 611 4- 22 58 __+ 2.5 111 4- 4.5 384 __+ 18 11.4 4- 0.6 6.9 ___ 0.3 DMBA 12 164 -+- 3 178 4- 4 387 4- 25 484 4- 19 81 -+- ~.9 87 4-/~ .8 291 4- 30 13.0 __+ 0.9 7.7 ~ 0.8

12 Control 10 167 4- 3 193 ___ 4 415 ___ 35 599 ___ 47 59 4- 2.7 117 ___ 4.4 376 4- 23 10.9 4- 0.8 6.5 __+ 0.2 DMBA 10 166 • 3 176 • 6 816 ~ 29 ~3~ -4- 86 66 __+ 7.3 92 • 2.7 306 4- 35 9.6 • 0.3 6.9 + 0.3

15 Control 10 162 -+- 3 195 • 4 366 ___ 20 597 ___ 24 57 -+- 1.9 106 4- 4.5 345 4- 27 10.1 4- 0.6 7.3 4- 0.1 DMBA 10 166 ___ 2 163 -4- 11 232 -+- 35 ~34 -4- 18 68 • 6.5 99 __+ 9.4 274 ___ 41 8.8 ___ 0.4 7.0 __+ 0.6

20 Control 10 162 -+- 3 216 4- 5 342 + 16 598 -+- 36 58 -+- 1.6 116 + 5.5 408 ___ 22 10.2 • 0.4 7.4 ~ 0.7 DMBA 10 168 ___ 3 203 ___ 8 322 +_ 24 678 ___ 87 54 • 3.5 101 __+ 5.9 334 4- 41 9.5 -+- 0.4 7.9 ~ 0.4

25 Control 9 167 + 4 231 • 4 374 • 23 593 • 15 60 ___ 1.9 124 _--!-6.1 411 4- 22 10.8 __+ 0.5 7.4 • 0.1 DMBA 8 170 4- 3 224 ___ 7 321 ___ 21 622 ___ 55 72 ___ 6.1 104 + ~.5 354 ___ 26 10.6 ___ 0.3 8.1 4- 0.3 DMBA Hydro- 165 4- 2 212 • 3 371 4- 30 704 4- 89 57 • 2.7 105 -+ 8~ 356 4- 8 10.6 -+- 0.2 7.7 ___ 0.4 cortisone 7 Organ and body weights of rats after 7,12-dimethylbenz(a)anthracene (DMBA). Female Sprague-Dawley rats, age 50 days, were given a single feeding of DMBA (133 mg/kg) in sesame oil by gastric tube. Control rats were fed sesame oil alone. Values are ex- pressed as the mean -4- S.E. for each group. Values in italics are signific~mtly different from controls, P < 0.05.

0.50 and 0.57 in the 9 groups of control rats. On Days 6, 9, large dark granules. Selected smears stained for leukocyte alka- and 25 after DMBA these ratios were decreased to 0.47, 0.49 line phosphatase showed a marked decrease in the enzyme in and 0.46; on Days 1, 2, 3, 12, 15, and 20 they were 0.53, 0.53, the few remaining granulocytes, a marked contrast to high levels 0.54, 0.52, 0.61, and 0.50. Uterine/body weight ratios were con- observed in the smears from control rats. The lymphocytes sistently lower in all groups given DMBA but were lowest on were immature and leukocytoid. Downey Type I, II, and lII Days 6 and 9--1.41 and 1.63 versus 1.81 and 2.0 for the control cells were observed in all smears on D:~.3's 10 and 15 and Types groups. I and II were still present as late as Day 24. Both lymphoid and myeloid elements remained below the initial values ob- Experiment 2. served on Day 1, but recovery of the myeloeytie series was Serial Blood Counts after DMBA. In contrast to Experiment definitely slower. Thrombocytopenia was nearly as severe in 1, the rats in this experiment received a lower dose of DMBA this group given a lower dose of DMBA; again, platelet T~ (20 mg per rat or approximately 113 mg/kg body weight) and turned out to be 4-5 days. But overt signs of toxicity were not blood indices were determined on the same animals on Days as marked and mortality was less (15% versus 29%). Mean 1, 2, 5, 10, 15, 19, and 24 after administration of the carcino- body and organ weights were in agreement with those reported gen. Control rats fed 1 ml sesame oil were bled at the same for Experiment 1 on Day 25. time. Observations for this experiment are summarized in Table 3. On Days 1 and 2 the total leukocyte count for the two Experiment 8. groups of rats was not significantly different, but the total Distribution of DMBA-aH in Spleen, Bone Marrow and number of myeloeytie elements had increased appreciably on Plasma. Unpublished observations (Flesher and Sydnor) indi- Day 2 in the group given DMBA, a phenomenon observed in cate the concentration of radioactivity in tissues and blood rats immediately after radiation. Leukopenia was maximal by from Sprague-Dawley rats, age 50 days, is highest during the Day 5, at which time the number of myeloeytie elements had 24-hour period following the administration of DMBA-aH by decreased from 2080 to 360 per eu mm and the lymphocytes gastric tube; Hamilton and Jaeobson (16) have published simi- from 6500 to 4050. Again, the granuloeytes which remained lar observations for plasma. Five female rats, age 50 days, were were large with hypersegmented nuclei and blue cytoplasm, fed 133 mg 7,12-DMBA-:~H (specific activity 32 microeuries/ with replacement of normal azurophilic granulation by a few m~ po~ kilogram body weight through a No. 8 .~ ..... ~" catheter.

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Table 2

Erythrocyte Day G-6-PD Erythrocyte after No. of Platelets Leukocytes/ Hematocrit (units) b GSH (rag %)~ Serum acid DMBA rats X 10-3/cu mm eu mm (%) (Mean) (Mean) phosphatase (U) ~ 1 Control 7 955 ~ 59 a 9,500 + 705 46.2 ~ 0.34 DMBA 12 931 ___ 32 8,317 ___ 475 48.8 4- 0.66

2 Control 7 1,023 ___ 21 9,650 __+ 551 47.3 _ 0.63 DMBA 12 822 --,- 37 8,270 -~- 888 50.4 4- 0.77

3 Control 10 915 • 50 9,080 ___ 1,038 46.6 4- 0.44 22.8 72.4 1.62 4- 0.11 DMBA 12 807 __+ 42 6,920 4- 357 48.4 4- 0.63 21.8 70.4 1.73 +__ 0.11

6 Control 10 872 ___ 31 9,050 ___ 731 46.6 ~ 0.51 22.2 65.0 1.67 4- 0.07 DMBA 12 613 • ~0 2~5~ ~ 887 42.4 4- 0~9 22.8 68.6 120 +__ 0.1

9 Control 10 886 ~ 29 10,080 __+ 864 46.7 4- 0.49 20.1 72.0 2.13 4- 0.17 DMBA 12 172 --~ 28 3,108 -~ 478 39,3 -~- 0.92 20.6 80.4 1.45 • 0.06 Hydrocortisone 7 263 • 38 2,860 ~ 587 38.7 4- 2,34

I2 Control 10 882 ___ 24 9,445 -~- 499 46.6 • 0.70 25.5 67.6 2.54 -~- 0.11 DMBA 10 129 4- 42 8,431 ~ 567 35.4 4- 2.77 24.4 72.8 1.86 4- 0.15

15 Control i0 902 ___ 34 10,805 -~ 723 48.1 4- 0.66 22.6 77.2 2.23 4- 0.09 DMBA 10 146 ~ 52 4,120 • 51,~ 21.8 • 4.18 20.8 89.1 1.56 4- 020 20 Control 10 881 ~ 48 7,790 ~ 438 46.5 4- 0.44 27.1 78.8 1.80 4- 0.11 DMBA 10 b'84 -+- 62 6,840 ~- 655 36.6 4- 8.45 27.1 78.8 1.78 +__ 0.15

25 Control 9 878 ~ 29 8,751 -+- 516 48.0 4- 0.65 22.1 70.4 1.61 4- 0.08 DMBA 8 939 ___ 39 6,862 -4- ~87 ~6.l 4- 0.51 23.2 68.6 1.72 ___ 0.21 DMBA & Hydroeortisone 7 793 ___ 62 7,428 ~ 715 40.0 ~ 2.95 I-Iematologic indices of control and DMBA-treated rats. Female Sprague-J)awley rats, age 50 days, were fed 133 mg DMBA/kg in sesame oil by gastric tube. Control rats were fed sesame oil alone. Values in itali(~s are highly significant, P ~ 0.05. DMBA, 7,12-dimethylbenz (a) anthracene. a Mean __ S.E. b One unit glueose-6-phosphate dehydrogenase (G-6-PD) represents the change in one O.D. unit per gram of hemoglobin. c Mg reduced glutathione (GSH) per 100 ml blood. d One unit of enzyme is defined as the activity which liberates one millimole p-nitrophenol per hour.

Table 3 Day after Leukocytes/ Platelets __ Percentage Total lymphoid Total myeloid DMBA Group cu mm X 10-3/cu mm L N M E elements/cu mm elements/cu mm 1 Control 9,840 4- 598 a 888 + 63 87.0 10.0 1.8 1.2 8,753 -~ 557 1,087 ___ 136 DMBA 9,115 ___ 384 928 __+ 37 82.9 16.6 0.3 0.2 7,584 +__ 253 1,572 4- 262

2 Control 9,300 __+ 650 1,080 __+ 43 84.5 13.5 0.5 1.5 7,905 ~ 587 1,395 • 190 DMBA 8,600 ~- 163 825 +__ 13 75.2 23.7 0.6 0.5 6,519 +__ 22~ 2,081 ~ ~56

5 Control 10,700 4- 650 958 __+ 28 85.0 12.4 2.6 9,095 ___ 461 1,604 4- 323 DMBA 4,400 +_- 258 696 +__ 60 92.1 7.8 0.1 ~,052 4- 685 863 4- 82

10 Control 10,000 4- 550 888 • 16 88.6 10.2 1.2 8,980 _ 442 1,020 ___ 130 DMBA 5,100 4- 817 281 -+- 43 86.6 11.8 0.8 0.8 ~,~57 ~ 814 6~,8 4- 138

15 Control 10,300 ~ 680 906 ~ 41 76.6 22.2 1.2 7,890 • 428 2,410 _ 409 DMBA 7,150 -4- 25/~ 410 +_ 96 84.6 14.4 0.6 0.4 6,092 -+- 318 1,058 -+- 285

19 Control 10,700 -4- 741 968 4- 78 76.0 19.8 1.6 2.6 8,303 __+ 534 2,397 __+ 235 DMBA 7,000 -4- 2~0 737 • 136 86.6 11.4 1.0 1.0 6,122 • 254 868 ~ 188

24 Control 10,000 4- 145 874 ___ 41 72.6 23.6 1.6 2.2 7,420 • 362 2,580 4- 313 DMBA 7,300 +__ 312 797 __+ 92 83.3 13.6 2.0 1.0 6,227 • 235 1,073 ~ 163 SeriaUy observed platelet and leukocyte counts in peripheral blood from female Sprague-Dawley rats given a single feeding of 7,12- dinmthylbenz(a)anthracene (DMBA), 20 rag, in 1 ml sesame oil by gastric tube, age 50 days. Control rats were fed sesame oil alone. L, b'mphoeytes, X, neutrophils, M, monoeytes, and E, eosinophils. Values italicized are significantly different h'om controls, P = 0.05. a Mean ___ S.E.

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Twenty-four hours later, they were bled from the abdominal ments two control rats were fed sesame oil and two were given aorta into a heparinized syringe under ether anesthesia. The a single 20-rag dose of DMBA dissolved in sesame oil by gastric spleens were weighed and quickly frozen in liquid nitrogen. tube. Twenty-four hours later blood was collected from the Marrow was obtained from both femora of each rat, pooled, abdominal aortae into ACD tubes and stored at 4~ Leuko- and frozen. Both spleen and marrow were dried in vacuo, then cyte, platelet, and erythrocyte counts were determined on the ground to a fine powder. Aliquots of the pulverized samples stored samples each day for two weeks. Chart 1 shows that and of plasma were prepared for radioassay by a modification the decrease in cellular elements in blood from control and of the oxygen-flask technic similar to that described by Hamil- DMBA-treated rats is similar, ifidicating that formed elements ton 'rod Jaeobson (16). Radioactivity was determined in a in circulating blood are not highly susceptible to damage by Packard Tri-Carb liquid Scintillation Spectrometer. The aver- the carcinogen. age values for spleen and bone marrow, respectively, were 770 and 872 dpm/mg, and for plasma, 344,000 dpm/ml. (One Experiment 5. mieroeurie is equivalent to 2.2 • 106 dpm.) The latter value Effect of DMBA on Heme Synthesis by Rat Reticulocytes. is equivalent to 5 micrograms DMBA. The concentration of l~(,tieuloeytosis was developed by intraperitoneal injection of a I)MBA necessary to damage the retieuloendotheIial system of physiologic saline solution of phenylhydrazine (initial dose 13.2 the rat appears to be extremely low. rag, then 6.6 mg on two consecutive days). Methylene blue, 0.2 gin, was injected subcutaneously twice each day to prevent Experiment 4. metlwmoglobin formation which occurs in phenylhydrazine- Influence of DMBA on Stored Rat Blood. To determine if l r(,atc~d rats (unpublished observations). Sixty hours after the DMI~A exerts a direct toxic effect on circulating cellular ele- initial inj('etion, a retic uloeyte count was (lone on each rat. The animals were paired :leeording to the percent reticuloeytes t~6 9 developed, and one rat of each pair was given 15 nag of 7,12- DMBA in 1 ml sesame oil by gastric tube. The control rat. of the pair was given 1 ml sesame oil alone. Twenty-four hours x later all animals were anesthetized and bled from the abdominal aorta into heparinized syringes. An aliquot of blood from .oe, v --.... each animal was taken for hemoglobin and retieuloeyte determinations and the remainder was used for the study of ~gFe uptake i~z vitro. Ferrous citrate labeled with ~gFe, specific activity 18.7 me/mg of iron, was added to the whole blood sample m an amount equivalent to 1.1 me/mg (0.068 mg Fe) and allowed to incubate at 20~ At 52, 107, and 156 minutes, respectively, a 1-ml aliquot was removed and added to 10 ml X B ~l cold phosphate-buffered saline (4~ the cells were washed once. The packed cells were then hemolyzed by the rapid freeze- \._------.. thaw method and the hemolysate counted in a well-type scintillation counter. To deternfine the total dose of ~gFe, 1 ml of J ~ 6 \ whole blood was hemolyzed and counted. till I-- 5- The radioactivity of the erythrocytes from the two groups of animals are shown in Table 4. The percent uptake of ~gFe ..I 4- was not. significantly different in the two groups, and no methe- 13. moglobin was detected in any of the samples. Iron taken up by rat retieulocytes under these experimental conditions has been shown to be used almost immediately for hemoglobin synthesis T (1, 4). Under these experimental conditions there was no de- -~ tectable suppression of retieuloeytosis nor of hemoglobin syn- X thesis by DMBA. Phenylhydrazine induced splenomegaly, loss of body weight, and increase in adrenal weight (Table 5). I ! I_ I I I I / I I n- I 2 3 4 5 6 7 8 9 10 DISCUSSION

OAY DMBA, a hydrocarbon with high carcinogenic potency, will invariably induce breast cancer in Sprague-Dawley female rats Charr~ 1. Disappearance c.urve of erythrocytes, platelets, and hmkocytes in blood from female rats given a single feeding of when administered as a single 20-rag feeding (18). The obser- 7,12-dimelhylbenz(a)anlhra('ene (DMBA), 111 mg/kg, (broke~t vations presented in this report show that it also induces pan- li,cs) or sesame oil, 1 ml, (solid lb~es). Blood was obtained from eytoI)enia by causing a profound depression of hematopoietic the ab(tomina.1 aor(a 24 hours after feeding and stored at 10~ ~ln(:l lymphoid precursors. In contrast to hemorrhagic necrosis Bloocl (.ounts wet'c, determined etwh day from the stored blood. of adrenal glands, which is evident 48 hours after DMBA,

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Table 4

No. of Itgb Retieulocytes (%) 59Fe uptake (cpm/ml hemolysate) % Group rats (gin %) Initial 9 Final b 52 rain 107 min 156 rain Uptake c Control 5 14.7 29.0 29.1 689 782 916 18.4 • • • 4-45 4-26 • 4-0.7 DMBA 5 15.1 28.6 33.3 596 730 828 19.2 4-0.1 4-2.03 4-1.95 • • 4-55 4-1.7 Mean (4- S.E.) uptake of 59Fe by rat reticulocytes in Vitro 24 hours after 7,12-dimethylbenz(a)anthracene (DMBA). a Prior to DMBA. b 24 hours after administration of 15 mg DMBA by stomach tube. c Percent of dose at 156 minutes.

Table 5 DMBA, observed by King may be the consequence of route

DMBA Sesame Oil of administration. Of some interest is the similarity of the effects induced by Initial body weight 139 135 DMBA and those induced by ehlorambucil and busulfan. Final body weight 131 124 Spleen 1.074 1.157 Elson (11, 12), and Elson et al. (13) compared the effect of Thymus 0.185 0.185 radiation with busulfan and chlorambucil on the hematopoietic Adrenals [two] 0.067 0.066 system of rats. The initial reaction to radiation is depression Liver 5.62 5.40 of lymphoid elements followed by granulocytopenia and thrombocytopenia which are maximal between Days 2 and 8. Ite- Mean body and organ weight (gm) of female Sprague-Dawley rats injected with phenylhydrazine. DMBA, 7,12-dimethylbenz(a)- eovery begins between Days 8 and 10. This hematologic re- anthracene. sponse could be imitated almost exactly by the simultaneous administration of busulfan and ehlorambueil. According to Elson, the initial weight loss after irradiation corresponds to hemorrhagic lesions in skin, lymph nodes, thymus, and other lymphoid depression, and the second period of weight loss is tissues, occur late (9 to 15 days) and coincide with throm- associated with depression of cellular activity in bone marrow. boeytopenia. This hemorrhagic diathesis, on the other hand, The hematologic response and pattern of body weight changes is the consequence of a biochemical lesion which occurs very after DMBA closely resemble that described for radiation (3). soon (within 24 hours) after exposure to DMBA, although DMBA causes maturation arrest of cells at the proerythro- overt morphologie damage is not apparent so early. Pulse- blast, myeloblast, and megakaryoblast levels. It does not ap- labeling of blood cell precursors with thymidine after an intra- pear to have any influence on the formed elements in peripheral venous injection of DMBA should shed further light on the blood. No morphologie changes were observed in retieulum sequence of events which occur at this early period. Because cells (stem cells), and the rapid recovery of the marrow sug- DMBA-aH was administered by gastric tube and radioactivity gests that these cells are not highly vulnerable to damage determined 24 hours later, the precise concentration which in- by this compound. Apparently only the more actively pro- duces this effect is uncertain. Unpublished observations (Flesher liferating hematopoietie elements are sensitive to DMBA and Sydnor) on the distribution of DMBA-3H in other tissues damage, but whether the proerythroblast, myeloblast, and suggest that it is unlikely to exceed 10 micrograms/gm of tis- megakaryoblast have the same affinity for the carcinogen or sue at peak concentrations which occur 8 to 12 hours after are damaged as a consequence of a block in a common meta- feeding. bolic pathway cannot be deduced from these experiments. Earlier, Shubik and della Porta (24) observed hematopoietie Several mechanisms by which DMBA may induce hema- changes in mice treated with a single application of DMBA topoietic injury may be considered: (a) a metabolic regula- (400 mg/kg). Only one of ten mice survived longer than tory circuit in which the carcinogen acts as a regulator may eleven days, thereby precluding a sequential study in recovery be modified (17), (b) enzyme systems concerned with DNA from damage. Huggins, et al. fed 20 mg DMBA to female synthesis may be damaged directly, or (c) DNA may be dam- Sprague-Dawley rats and observed prolonged leukopenia and aged by intercalation of hydrocarbon between base pairs. hemorrhagic adrenal necrosis (18, 19). The adrenal glands on Heidelberger (17) believes it is unlikely that interaction with Day 3 contained approximately 20 times the amount of hemo- DNA is an important mechanism. Brookes and Lawley (7), globin found in untreated control rats. More recently, King (21) reported depression of hematopoiesis in male albino rats on the other hand, have presented evidence that DMBA, or following the application of DMBA (64 mg) dissolved in olive a yet unidentified metabolite of DMBA, is bound to DNA oil to skin. Changes in hematopoiesis were not as profound, and that maximal binding occurs during the initial 24-hour but the sequence was similar to those presented in this paper. period after treatment. These investigators have suggested Since the pattern of hematopoietie depression and recovery that DNA may be the essential cellular receptor of the ear- following the administration of toxic agents is dependent on einogen. The observations of Jensen et al. (20) that DMBA dose and route of administration, the less severe and more depresses incorporation of tritium-labeled thymidine in the variable hematopoietie response, despite a higher dose of perchloric acid-insoluble fraction of rat testis, intestine, and

326 CANCER RESEARCH VOL. 28

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1968 American Association for Cancer Research. DMBA Suppression of Cellular Activity in the Rat adrenal further support the concept that DNA is affected. Radiomimetic Chemicals on the Blood. Brit. J. Haematol., 1: Although direct evidence is lacking, it is suggested that DMBA 104-116, 1955. affects the hematopoietic system by inhibiting DNA replica- 13. Elson, L. A., Galton, D. A. G., and Till, M. The Action of tion. Chlorambucil (CB-1348) and Busulfan (Myleran) on the Haematopoietic Organs of the Rat. Brit. J. Haematol., 4: 355- (differentiation) 374, 1958. STEM CELL 9 ,-MATURE CELL 14. Evelyn, Kenneth A., and Malloy, Helga T. Microdetermina- (no injury) T (no injury) tion of Oxyhemoglobin, Methemoglobin and Sulfhemoglobin injury here in a Single Sample of Blood. J. Biol. Chem., 126: 655-662, 1938. The close resemblance of the sequence of events observed after 15. Feinendegen, L. E., Odartchenko, N., Cottier, H., and Bond, DMBA to those reported for experimental animals treated by V. P. Kinetics of Megacaryocyte Proliferation. Prec. Soc. radiation or alkylating agents, both of which mediate their Exptl. Biol. Med., 111: 177-182, 1962. effects by damaging DNA, could be coincidental. But this 16. Hamilton, T., and Jacobsen, H. I. Distribution of Radioac- seems unlikely. The morphologic changes observed in the mar- tivity in Plasma after Feeding Tritiated 7,12-Dimethylbenz- (a)anthracene. Prec. See. Exptl. Biol. Med., 118: 827-829, row from DMBA-treated rats are quite similar to those ob- 1965. served in other disturbances of DNA replication, e.g., vitamin 17. Heidelberger, C. Studies on the Molecular Mechanism of Hy- B12 deficiency in which there is a failure of thymidylate syn- drocarbon Carcinogenesis. J. Cellular Comp. Physiol., 64 thetase. (Suppl. 1): 129-148, 1964. 18. I-Iuggins, C., Grand, L. C., and Brillantes, F. P. Mammary REFERENCES Cancer Induced by a Single Feeding of Polynuelear Hydro- carbons and Its Suppression. Nature, 189: 204-207, 1961. 1. Allen, David W., and Jandl, James H. Kinetics of Intracellu- 19. Huggins, C., and Morii, Sotokiehi. Selective Adrenal Necro- lar Iron in Rabbit Reticuloeytes. Blood, 15: 71-81, 1960. sis and Apoplexy Induced by 7,12-Dimethylbenz(a)anthra- 2. Anderseh, M. A., and Szezypinski, A. J. Use of p-Nitrophenyl- cene. J. Exptl. Med., 11/~: 741-760, 1961. phosphate as the Substrate in the Determination of Serum 20. Jensen, E. V., Ford, E., and Huggins, C. Depressed Incorpora- Acid Phosphatase. Amer. J. Clin. Pathol., 17: 571-574, 1947. tion of Thymidine=aH into Deoxyribonucleic Acid Following 3. Baeq, Z. M., and Alexander, P. Chemicals which Simulate the Administration of 7,12-Dimethylbenz(a) anthracene. Prec. Biological Effects of Ionizing Radiations. In: Z. M. Baeq and Natl. Acad. Sci. U. S., 50: 454~t59, 1963. P. Alexander (eds.), Fundamentals of Radiobiology, Ed. 2, pp. 21. King, M. A. R. The Acute Effects of the Carcinogenic Hydro- 217-238. London: Pergamon Press, 1961. carbon, 7,12-Dimethylbenz(a)anthraeene on the Hematopoie-

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1968 American Association for Cancer Research. Suppression of Cellular Activity in the Reticuloendothelial System of the Rat by 7,12-Dimethylbenz(a)anthracene

Madison J. Cawein and Katherine L. Sydnor

Cancer Res 1968;28:320-327.

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