The effect of repeated doses of urethane (ethyl carbamate) on the mitotic activity and cellular composition of the bone marrow of normal mice

A single dose of urethane produced a rise in the mitotic index, due essentiallyto a large increase in the metaphase index in the bone marrow of normal mice.Repeated daily injections (three and six doses) produced an elevation of themitotic index, which was, however, smaller than that observed after a singledose of urethane. After nine doses of urethane the mitotic index was found approximately at the same level as in the controls. The mitotic index in the bonemarrow of urethane-treated mice never decreased below the normal level foundin untreated mice.

After three, six and nine doses of urethane a rise in the percentage of ana- andtelophases was found, in contrast to the increase in the percentage of metaphasesafter administration of a single dose of urethane. The percentage of prophaseswas decreased after six and nine doses of urethane.

The morphological changes in the mitotic figures were the same as thosedescribed after a single dose of urethane. They consisted mainly in bridge formation and chromosome aberrations in anaphase.

Very large promyelocytes, often with prematurely lobated or bizarre shapednuclei, were observed.

There was damage to the nuclei of the erythroid precursors and consequentlyan increase in the myeloid-erythroid ratio in the bone marrow after repeateddoses of urethane, especially after nine doses.

The maturation rate of the segmented leukocytes in the bone marrow of theurethane treated mice was augmented after repeated doses.

Submitted on February 27, 1956 Accepted on June 25, 1956     

Hematologic effects of regional nitrogen mustard therapy

1. The hematologic findings on 21 patients who received fractionated intra-arterial HN₂ therapy are presented. The arteries cannulized were the externalcarotid in 9 cases, the internal carotid in 4 cases, the external iliac in one caseand the lower abdominal aorta in 7 cases. The total dose of drug given variedfrom 0.25 to 3.10 mg./Kg.

2. The effects on bone marrow and peripheral blood were essentially the samein character as those observed after intravenous administration of HN₂. Largerdoses of HN₂ were tolerated when administration was intra-arterial.

3. A consistent early depression of marrow eosinophils was noted in all caseswith original differential counts of 2 per cent or more, except in one patient whohad Hodgkin’s disease and extensive psoriasis.

4. A differential effect of HN₂ on marrow in the region of therapy and themarrow from other sites was demonstrated by simultaneous aspiration studies.

5. Factors reducing hematologic damage include occlusion of venous returnfrom the injected site and prolongation of therapy.

6. Only one patient in this group developed severe hypoplasia of the bonemarrow. Studies on this and other patients treated by the lower abdominal aorticroute suggest that more than 1.1 mg./Kg. HN₂ per course in this region may behazardous.

7. Four patients died within two weeks following therapy with symptomssuggesting profound electrolyte disturbance. It is probable that limiting factorsother than hematologic may occur with localized HN₂ therapy.

     

Acute thrombocytopenia induced in dogs by administration of urethane (ethylcarbamate)

1. Dogs treated for one to two weeks with daily injections of urethane (ethylcarbamate) subcutaneously, in doses of 0.4 Gm. per Kg. of body weight, presenteda typical picture of thrombocytopenic purpura.

2. The pathologic changes consisted in numerous purpuric lesions in the smallintestine and a smaller number on the skin, heart, lung, cortical zone of the kidney,epiploon, and, rarely, on the stomach and large intestine.

3. The hematologic changes occurred in the following sequence: leukocytosis,leukopenia and thrombocytopenia, and finally acute anemia coinciding withsevere intestinal hemorrhage. In the final phase, the coagulation time was normaland the bleeding time very much increased.

     

THE HEMATOLOGIC RESPONSE IN DOGS TO THE ADMINISTRATION OF ANTI SPLEEN SERUM

1. Intravenous injection into dogs of rabbit anti-dog-spleen serum in doses of0.04 cc./10 Kg. results in a significant increase in the mononuclear cells of theperipheral blood.

2. The rise in circulating mononuclears occurs promptly, with 24-48 hours, andis sustained, generally for 20 days.

3. A large dose of the same serum, 5.4 cc./10 Kg., exerted an opposite effect,poducing a significant decrease in circulating mononuclears.

4. The possible significance of these findings is discussed.

     

Megaloblastosis Produced by a Cytosine Antagonist: 1-beta-D-arabinofuranosylcytosine

1. Cytosine arabinoside induced objective, but temporary, decrease of tumormasses in three patients with lymphosarcoma and slight decrease in some lesions in two out of ten treated patients with disseminated carcinomatosis.

2. In doses of 3 to 50 mg./Kg. given at varying intervals, cytosine arabinosideinduced definite megaloblastic changes in the marrow of all patients studied.Mitotic abnormalities similar to those found in other megaloblastic anemiasalso occurred.

3. Associated with bone marrow changes, depressions of hemoglobin, whiteblood cells and platelets in the peripheral blood were observed.

4. The exact mechanism of action of cytosine arabinoside has not beenelucidated. It is speculated that because of the close structural similarity between cytidylic acid, cytosine arabinoside could interfere with DNA synthesis.

Submitted on August 6, 1962 Accepted on November 7, 1962     

Volume and Cellular Constitution of Bone Marrow in Guinea Pigs Hypoxic from Birth

A quantitative study of the volume and cellular constitution of the bonemarrow was carried out in 10 guinea pigs of approximately 400 Gm. bodyweight, which had been born and reared at a simulated altitude of 14,000 feet,and in 10 normal controls.

The hemopoietic marrow of the experimental group was more cellular andshowed a marked increase in the number of erythroid cells per unit volume.No significant changes were demonstrated in the absolute counts of othercells, but fat vacuoles appeared less numerous. The fatty (yellow) marrowoccupied its normal centrifugal position in the skeletons of both groups.

No absolute increase in the volume of bone marrow was detected in theexperimental group, but the red marrow volume formed a significantly greaterproportion both of the volume of the skeleton containing it and of the totalmarrow volume.

It was concluded that the marrow of the hypoxic animals had a total population of erythroid cells about 40 to 50 per cent greater than normal; this increasewas largely accommodated by closer packing of cells in the red marrow.

Submitted on October 23, 1959 Accepted on November 20, 1959     

Hematologic Changes in Rat Following Administration of Desoxypyridoxine, Aminopterin, Oxythiamine and 6,7-Dimethyl-9-(2'-acetoxyethyl)isoalloxazine

1. Administration of aminopterin to rats at a level of 100 /Kg./day for a10 day period produced leukopenia which was rapid in onset and led quicklyto death, whereas the leukopenia produced by the administration of desoxypyridoxine at the level of 40 mg./Kg./day for a 2 week period or at 100mg./Kg./day for a 4 week period was not accompanied by toxic effects suchas loss of appetite, diarrhea and hemorrhage and was characterized by lymphocytopenia and granulocytosis.

2. The results of combining the administration of aminopterin (70 /Kg./day) with desoxypyridoxine (40 mg./Kg./day) for a 2 week period indicatedan additive effect of the two in the production of lymphocytopenia; thegranulocytosis resulting from desoxypyridoxine administration was counter-acted by the granulocytopenic effect of aminopterin. Also, the leukopeniaproduced by the combined administration of aminopterin and desoxypyridoxine was not accompanied by the toxic effects which follow aminopterinadministration.

3. Oxythiamine alone or in combination with desoxypyridoxine or aminopterin did not influence the leukocyte count.

4. Administration of aminopterin with 6, 7-dimethyl-9-(2'-acetoxyethyl)isoalloxazine (U-2112) produced marked lymphocytopenia and granulocytopenia accompanied by toxic effects such as loss of appetite, diarrhea and hemorrhage.

See PDF for Table

Submitted on January 31, 1959 Accepted on March 9, 1958     

Autologous bone marrow infusion as an adjunct in therapy of malignant disease

1. Three patients with lymphosarcomatosis and two with disseminatedHodgkin’s disease were treated with massive doses (super-dosage) of nitrogen mustard (over 1.0 mg./Kg.) given intravenously, followed by the intravenous infusion of autologous bone marrow.

2. Two of the five patients succumbed to infection. The remaining threepatients recovered within one month and experienced brief remissions.

3. It is suggested that this form of therapy merits further trial. Thus, itmay be considered for use earlier in the course of the disease when thereis a smaller total amount of tumor tissue, and thus a greater possibility forits complete eradication. Its use in other neoplastic disorders, as followingoperative procedures disclosing malignancy, is suggested.

Submitted on July 25, 1958 Accepted on September 26, 1958     

Urethane therapy in leukemia

1. The results of urethane therapy in 24 cases of leukemia are described.

2. The average daily dose is 4 Gm. orally or intravenously.

3. The drug is irregularly effective. Chronic myelogenous leukemia appears moreresponsive than the lymphatic variety.

4. Acute leukemias are not significantly altered in course by urethane.

5. Urethane produces a fall in the total leukocyte count in a majority of all typesof leukemia. Clinical improvement does not necessarily follow.

6. Nausea is the most frequent side effect of urethane therapy. Possible marrowaplasia is the most dangerous toxic effect.

7. Urethane is of definite, but of limited value in the treatment of chronic leukemia. In some instances, it compares favorably with x-ray therapy, but in general,it is less dependable, particularly in its frequent failure to induce optimum returnof normal red cell and platelet values, and optimum regression of organ infiltration.

     

Leukokinetic Studies. VII. Morphology of the Bone Marrow and Blood of Dogs Given Vinblastine Sulfate

The effect of a single injection of vinblastine sulfate was studied in 50mongrel dogs. Nine of 34 dogs given 0.2 mg./Kg. of VLB died with gastrointestinal toxicity and the mortality rate increased as the dosage of VLB wasincreased. The morphologic pattern of leukocyte suppression and recovery inthe bone marrow and blood was studied in detail in surviving animals.

The cells of the bone marrow were markedly affected by VLB. Within 4hours there was an increase in the number of cells in metaphase and, by day1, virtually all proliferating leukocytes and erythrocytes had disappeared. Anorderly repopulation of the bone marrow followed.

The neutrophils, eosinophils, lymphocytes and monocytes of the blood wereall markedly altered in concentration after VLB. Each type of cell first decreased to abnormally small numbers and then increased to abnormally largenumbers in the blood. The curve of disappearance from and reappearance inthe blood differed for each cell type.

The changes in blood neutrophil number and morphology were correlatedwith changes in the blood neutrophil precursor cells of the marrow. The following conclusions were reached concerning the neutrophils and the assumptions implicit to these conclusions were detailed.

1. In the dog, the marrow contains enough post-mitotic granulocytes toreplace those lost from the blood for at least 3 to 4 days.

2. The release of mature neutrophils from the bone marrow is a functionof the rate at which blood neutrophils are lost and proceeds normally evenwhen the marrow granulocyte reserve is partially depleted.

Submitted on March 27, 1963 Accepted on August 20, 1963     

Biochemical Lesion in Dilantin-Induced Erythroid Aplasia

Clinical and biochemical studies are reported in a patient who developedpure red cell aplasia after 2 years therapy with Dilantin but in whom aplasiacould subsequently be induced with 4-5 Gm. of the drug. Recovery occurredregularly when the drug was discontinued.

The administration of 1500 ml. of patient’s plasma obtained during theaplastic phase to a normal volunteer with Dilantin had no effect on the recipient’s reticulocyte count, number of nucleated red cells in his bone marrow,or Fe⁵⁹ clearance. Attempts to demonstrate binding of gamma globulin tothe patient’s nucleated red cells in the presence of Dilantin by immunofluorescence technic were unsuccessful.

Dilantin, in a concentration of 20 µg./ml. in vitro, caused significant inhibition of the uptake of C¹⁴ formate, glycine, adenine, orotic acid, and uridine intoDNA but not into RNA of patient’s bone marrow studied when fully recovered. There was no effect on the uptake of deoxyuridine or thymidine.

The Dilantin effect was specific to the erythroid cells as shown by radioautographic studies and by the absence of inhibition when these cells wereabsent from the bone marrow.

Vitamin B₁₂ and folinic aci dgiven to the patient in large doses did notreverse the hematologic effects of Dilantin. The daily intravenous administration of all four deoxyribonucleosides, deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine had a suggestive, but not clear-cut, effect.

In an effort to examine a possible relationship of the pure red cell aplasiain this patient to riboflavin metabolism, the patient was started simultaneouslyon Dilantin and riboflavin. Dilantin was now without effect. However, thepatient remains refractory to Dilantin a year after riboflavin was discontinued.

It is concluded that Dilantin exerted its toxic effect in this patient byspecifically inhibiting DNA synthesis in erythroid cells probably at the stepof deoxyribotide formation.

The role of riboflavin in the development of resistance to the toxic effect ofDilantin in this patient remains uncertain.

Submitted on January 19, 1967 Accepted on May 5, 1967     

Brief Note: Tritiated Thymidine as a Cytocidal Agent in Human Leukemia

1. Two patients with acute leukemia had considerable decreases in leukemiccells in the peripheral blood as well as reduction in size of spleen and leukemicmasses after 10 injections of ³H-TDR given over a 5-day period. Each injectionwas 0.25 µc./Gm. body weight.

2. The pertinent aspects of cytotoxic effects of ³H-TDR are reviewed.

3. The radiation doses delivered to the nucleus are estimated from autoradiographic data.

4. Evidence is presented for the observed effects being due to ³H-TDR.

Submitted on July 19, 1965 Accepted on May 10, 1966     

Studies on Erythropoietic Action of Angiotensin II

The effects of synthetic Angiotensin II on erythropoiesis were investigated.

1. Two daily intravenous injections of Angiotensin II, 100 µg./Kg. of bodyweight, revealed no acceleration of Fe⁵⁹ incorporation into erythrocytes ofeither normal rabbits or hypophysectomized rats.

2. When given by intravenous drip, 100 µg./Kg. of Angiotensin II significantly accelerated the radioiron incorporation.

3. The renal blood flow was markedly reduced throughout the period ofthe dropwise injection; the same effect was transitory after single intravenousinjection. The elevation of plasma erythropoietin activity was observed inhypertransfused polycythemic rabbits following the dropwise injection ofAngiotensin II.

From these results, it is concluded that erythropoietic activity of AngiotensinII results from an increased erythropoietin production. The increased production may have been induced by a renal ischemia through administration ofAngiotensin II.

Submitted on February 23, 1966 Accepted on January 13, 1967     

Comparative influence of ouabain,norepinephrine and heart rate on myocardial oxygen consumption and inotropic state in dogs

The purpose of this study was to compare the myocardial oxygen cost of augmented inotropic state produced by ouabain, norepinephrine, or increased heart rate. This problem was examined in dogs using an isovolumically contracting left ventricular preparation. Inotropic state was measured as the maximum observed contractile element velocity at the lowest common level of wall stress (MAX V). Peak left ventricular wall stress was maintained constant in each dog so that it would not influence changes in myocardial oxygen consumption (MVO₂). Ouabain (4 × 10² μmoles/Kg.) and norepinephrine (2 × 10³ μmoles/Kg./minute) always augmented inotropic state (MAX V) and increased MVO₂. The positive slopes of the regression of MVO₂ on MAX V for ouabain (45.4 ± 12.5 μl/beat/100 Gm./muscle length/sec; mean ± SEM) and norepinephrine (34.5 ± 5.6 μl/beat/100 Gm./muscle length/sec; mean ± SEM) were not significantly different, indicating that for an equal augmentation of inotropic state, ouabain increases myocardial oxygen demands to the same extent as does norepinephrine. When the results with ouabain or norepinephrine were compared to results obtained by altering heart rate, it was found that increasing inotropic state by these pharmacologic agents is more costly in terms of myocardial energy demands than when inotropic state is enhanced by increasing heart rate.     

An Attempt to Produce Hypersplenism in the Dog, Using Methylcellulose

(1) Hematological and histopathologic changes were studied in dogs andrats after infusions of solutions of methylcellulose.

(2) After 4 to 8 weeks of daily intravenous infusions of 0.6 Gm. of 400 centipoise methylcellulose, the dogs developed moderate anemia with a reductionof the mean red cell volume to 27 ± 4 cc./Kg., compared with 38.6 ± 3 cc./Kg.in the controls, and a reduction in the apparent red cell life span studied withCr⁵¹ from 24 ± 3 days in the controls to 18 ± 3 in the treated animals.

(3) Following the infusions in the dog there was an increase of the meanblood urea nitrogen from 15 mg. per cent to 96 mg. per cent, with only a smallincrease in the rat.

(4) At necropsy there was foamy cytoplasmic vacuolization throughout thereticuloendothelial systems of both rat and dog. In the dog, the spleens werepale and moderately enlarged. Large cells with vacuolized cytoplasm largelyreplaced the normal structures of this organ. In the markedly enlarged ratspleen there were islands of methylcellulose-filled cells, surrounded by a rimof lymphocytes and strikingly engorged red pulp.

In the rat there was vacuolization of the renal glomerular cells but no significant change in the tubules or interstitial tissue. In the dogs sacrificed oneweek after the termination of the methylcellulose infusions, foamy cytoplasmicdistention of the glomeruli and interstitial cells and tubular dilatation and cystformation were noted. Progressive fibrosis and hyalinization of the glomerulusand fibrosis and calcification and lymphocytic infiltration of the interstitialtissue were seen in the kidneys of dogs sacrificed from 3 months to 3 years aftertermination of the methylcellulose infusions.

(5) Although methylcellulose infusions produce splenomegaly and anemiain the dog, the associated uremia precludes this preparation as a model forthe study of hypersplenism.

Accepted on February 23, 1961     

The Colchicine Method in the Study of Bone Marrow Cell Proliferation

The rate of metaphase accumulation in the bone marrow cells of AKR micetreated with colchicine was investigated. The influence of this alkaloid onthe differential count of the bone marrow cells in these animals was alsostudied. It was demonstrated that the stathmokinetic effect of colchicine onthe bone marrow cells started almost immediately after the administration ofthe drug. The number of arrested metaphases increased linearly from one-halfhour to six hours after the injection of colchicine, and then fell rapidly.

In rats injected with colchicine, the changes in the bone marrow concentration of this compound were followed for eight hours. The colchicine concentrations increased from the first to the fourth hour, and then fell rapidly, reachingthe zero level at the eighth hour.

From the results obtained, it appeared that four hours post-injection wasthe most convenient time for the study of the bone marrow proliferative activity by the colchicine method when 1.2 mg./Kg. of colchicine per body weightwas used. The fact that four hours after the injection, a mild decrease in thepercentage of mature bone marrow granulocytes was found, may represent alimiting factor, which is, however, of moderate importance in the application ofthis technic.

Submitted on May 5, 1961 Accepted on June 14, 1961     

Protective Effect of Leukapheresis in Man Against Large Dose Antineoplastic Chemotherapy

Eight patients with widespread metastatic neoplastic diseases followingleukopoietic stimulation by leukapheresis were treated on 10 occasions withlarge doses of HN2, 30 to 280 mg. (0.6 to 4.1 mg./Kg.). Despite severe hematodepressionwith anemia, leukopenia and thrombocytopenia, hematopoieticrecovery occurred in all instances within 16 to 25 days. The recovery fromsevere hematodepressant doses of HN2 is attributed largely to the previousleukopoietic stimulation. Doses of HN2 at approximately four times the LD₅₀were unable to eradicate metastatic tissue in Ewings sarcoma and malignantmelanoma.

Although hematopoietic protection was evident, the hazard of injuring thenext most vulnerable tissues must be considered as larger amounts of cytotoxicagents are employed.

Submitted on May 4, 1959 Accepted on May 31, 1960     

Protein Synthesis in Rat Lymphocytes: Radioautographic Studies of Availability and Utilization of Labeled Amino Acids in Vivo

Injections of H³-methionine and H³-leucine were combined with radiochemical and radioautographic technics to study the availability time ofH³-methionine and the protein synthetic ability of rat lymphocytes in vivo.

Although 98.5 per cent of H³-methionine was removed from the serum5 minutes after injection, sufficient quantities persisted and/or re-entered theserum from tissues to cause increasing grain counts in radioautographs oflarge lymphocytes for 1 hour after isotope administration. A small amount ofadditional labeling occurred during the 2nd hour, but it is calculated thatlabeling is 97-98 per cent complete by 1 hour.

All of the large and medium lymphocytes were labeled in the thymus, lymphnode, and thoracic duct lymph at short intervals after injection of 4 µc./Gm.body weight of H³-methionine. Evidence is presented that protein synthesisoccurs in the nucleus as well as in the cytoplasm and that newly formed protein is equally distributed between daughter cells following mitosis. Previousimmunochemical studies are combined with information on generation timeand disappearance rates of radioactivity to suggest that large and mediumlymphocytes are constantly producing and releasing proteins. Large andmedium cells in lymph and lymph node are more active in this than aresimilar cells in the thymus. Evidence of reutilization of labeled metabolitesin the lymph node and especially in the thymus is discussed.

Although not all small lymphocytes were labeled by 4 µc./Gm. body weightof H³-methionine, it was shown that larger doses of isotope would label 100per cent of them. Small lymphocytes in thoracic duct lymph evidenced significant turnover of labeled protein during the 1st day after isotope administration.

Submitted on August 21, 1963 Accepted on November 9, 1963     

Leukapheresis in Man. III. Hematologic Observations in Patients with Leukemia and Myeloid Metaplasia

Eleven patients with leukemia and one patient with myeloid metaplasiaunderwent leukapheresis on 18 occasions for 94 to 275 minutes during which93 to 1668 x 10⁹ leukocytes were removed. All patients exhibited a significantand continued decline of peripheral leukocyte concentration during or afterthe procedure. In 12 of the 18 instances, the leukocyte concentration returnedslowly to the initial leukocyte level within 1 hour to 22 days. The numberof leukocytes withdrawn represented 16 to 247 per cent of the initial circulatingvolume removed at a rate of 0.13 to 1.14 leukocyte blood volumes per hour.The RAR was 1:1 to 12:1 to the circulating leukocyte number. Rate of replenishment of the circulating immature leukocyte numbers were 4.0 x 10⁷to 52.2 x 10⁹/Kg./day. The PMN’s were replaced at rates of 10 x 10⁶ to 7.05x 10⁹/Kg./day which were equal to or slower than in normal subjects. Changesin number occurred in the dominant leukemic cell types without significantshifts in the differential counts. No changes in marrow population other thana slight decrease in cellularity were observed.

The data indicate that in the leukemic patient the peripheral leukocyte concentration was not maintained or replenished promptly following the withdrawal of sizeable quantities of leukocytes, demonstrating a block in transferof leukocytes from the tissues to the blood. This is in marked contrast to theleukocytosis and marrow stimulation observed in hematopoietically normalsubjects following leukaphereses.

The platelet counts fell promptly during leukapheresis, returning towardcontrol levels in eight studies within 7 hours following the procedure. In fourstudies the platelet counts returned to control levels in 3 to 9 days. Thechanges in platelet concentrations were similar to those observed withhematologically normal subjects. The size of the platelet reservoir in theseleukemic patients is about twice that of the circulating blood.

Submitted on May 14, 1962 Accepted on October 14, 1962     

Massive Nitrogen Mustard Therapy in Hodgkin's Disease with Protection of Bone Marrow by Tourniquets

1. Eight patients with far advanced Hodgkin’s disease were treated withmassive single doses of nitrogen mustard (0.95-1.5 mg. /Kg.) with tumor regression in each case.

2. An attempt was made to protect the bone marrow of the extremities fromthe effects of the nitrogen mustard by applying orthopedic tourniquets duringthe injection. There was definite evidence that the marrow was protected bythis procedure. Marrow in the tibia became progressively more cellular duringthe time that the marrow in the torso degenerated to almost complete aplasia.During the period of recovery, radioactive iron was given and more of itlocalized in the protected arm than in the unprotected, evidence of greatererythropoietic activity.

3. Neurologic complications occurred at the doses employed.

4. Serious gastrointestinal pathology was not demonstrated at these doses.

5. Prior extensive radiation therapy prolonged the hemopoietic depressiondue to nitrogen mustard. This was not produced by previous mustard therapy.

6. Color change in ecchymotic areas may be used to predict bone marrowrecovery.

7. Dividing the massive dose seems to result in less toxicity.

Submitted on December 14, 1959 Accepted on March 8, 1960