Autoradiographic Studies of the Action of Leucogenenol on the Blood Cells of The Rat

Abstract

Leucogenenol, a compound isolated from the metabolic products of Penicillium gilmanii by Rice (1) and from human and bovine liver by Rice and Shaikh ( 2 ) and indicated by degradation studies to be 2-( 1, 2-dihydroxy-3-methyl-5-oxo-cyclohexyl) -3 , 1 1dihydroxy-11 -(hydroxymethyl) -9-methyl-loxa-5-azaspiro [ 5,s ] undeca-2,4-diene-7-one (3), induces a leukocytosis when it is injected intravenously or intraperitoneally into rabbits (4) , dogs, mice, or monkeys (5,6).Injection of leucogenenol into animals is followed by a relative increase in the number of myeloblasts in the bone marrow and lymphoblasts in the spleen ( 5 , 6), suggesting that leucogenenol stimulates the rate of replication of these cells from their respective precursors. The stimulatory action of Ieucogenenol on lymphoid cells is also indicated by the finding that leucogenenol decreases the latent period required for antibody production in sublethally X-radiated mice (7). The addition of leucogenenol to a culture of human lymphoblastoid or mouse leukemic cells increases their respiratory quotient and their rate of replication (8), suggesting that leucogenenol acts directly on the blood cells of animals.I t was of interest, therefore, to establish whether or not leucogenenol acted specifically on one or more of the progenitors to the circulating blood cell. Patt and Maloney demonstrated (9) that tritiated thymidine is first incorporated in the blast forms of myeloid and erythroid cells and then is carried through the series of myeloid and erythroid cells to appear in the corresponding circulat-ing cells approximately 24 hr later. It appeared therefore that studies of the effect of leucogenenol on the time and intensity of the labeling of bone marrow blood cells with tri t ia ted thymidine should indicate whether or not leucogenenol acted on one or more types of myeloid and erythroid blood cells. SimilarIy it might be possible to establish whether or not leucogenenol acted on one or more types of lymphoid cells. Accordingly rats were injected with tritiated thymidine and leucogenenol and the type and number of labeled blood cells in their bone marrow, spleen, and peripheral circulation compared by standard autoradiographic techniques (10-12) with the number of corresponding labeled cells in the bone marrow, spleen, and peripheral circulation of rats that were injected with tritiated thymidine alone.Materials and Methods. Each of 60 rats (Wistar, approx. 250 g) was injected intraperitoneally with tritiated thymidine2 (1 pCi/g of body wt). At approximately the same time 30 of the rats were injected intravenously through the tail vein with 0.02 pg of the calcium salt of leucogenenol dissolved in 0.2 ml of pyrogen-free isotonic saline. At intervals of 6, 12, 24, 48, and 96 hr thereafter a sample of blood was obtained from the tail vein of each rat, diluted in the usual manner in a Trenner pipet and the number of leukocytes was determined by means of a hemocytometer. Counts made in duplicate differed by less than 10%. Smears of blood for differential coun...

The Action of Leucogenenol on Lymphoblastoid Cells of Normal and Neoplastic Origin

“…However lymphoblastoid cells of neoplastic origin may be characterized by the RQ and rate of oxygen consumption and the magnitude of the effect of leucogenenol on the RQ and rate of oxygen consumption. The fact that leucogenenol causes an increased rate of maturation (5,6) of the progenitors of the peripheral leukocytes and also increases blast formation of human peripheral leukocytes in tissue culture (11) suggests the possibility that leucogenenol acts on an enzyme system that in tissue culture results in a change in RQ and oxygen consumption, while in the animal the same system is associated with the maturation or transformation of blood cells. The uniformity of action of leucogenenol on lymphoblastoid cells of normal human origin compared to the variable and characteristic action of leucogenenol on lymphoblastoid cells of neoplastic origin suggests the possibility that malfunctioning of the system that is affected by leucogenenol is responsible for neoplastic blood diseases.…”

“…Within 24 hr after the injection of leucogenenol, granuloblasts and mlore immature cells are provided with higher than normal proliferation and maturation rates ( 5 ) ; and, consequently, injection of irradiated recipients with bone marrow from mice previously injected with leucogenenol causes an increased rate of colony formation in the spleens of the recipients ( 5 ) . The maturation rate of each of the morphologically recognizable progenitors of the circulating neutrophil and erythrocyte is increased by leucogenenol (6) and neutrophils are released into the circulation to be later sequestered by such tissues as the spleen (7). The proliferation and maturation rate of lymphocytic cells is also increased (6, 7) by leucogenenol ; and, consequently, there is a decrease in the time required for sublethally irradiated (8) and splenectomized (9) mice and rats to become capable of forming hemolysin antibodies.…”

Effect of Leucogenenol on Human Peripheral Lymphocyte Cultures

“…In tissue cultures of lymphoblastoid cells the preparation causes a marked change in the RQ as well as the rate of replication of the cells [14], increases the incorporation of DNA in the primitive proliferat ing pool of cells [15] and stimulates the rate of formation of lymphocytes from a precursor [16]. By degradation studies [17] leucogenenol was be lieved to be 2-(l, 2-dihydroxy-3-methyl-5-oxocyclohexyl)-3,11-dihydroxyll-(hydroxymethyl)-9-methyl-l-oxa-5-azaspiro (5,5) undeca-2,4-diene-7-one.…”