Document DM68Lg03NZwv3wO55Xqn6j0RO

FUNDAMENTALS OF TOXICOLOGIC PATHOLOGY Editors: Wanda M. Haschek-Hock University of Illinois Colin G. Rousseaux University of Saskatchewan CHAPTER 16 BLOOD AND BONE MARROW RICHARD D. IRONS, M.T., Ph.D. Molecular Toxicology and Environmental Health Sciences Program School of Pharmacy and Department of Pathology School of Medicine University of Colorado Health Sciences Center, C-235 4200 E. 9th Avenue Denver, Colorado 80262 ACADEMIC PRESS, IN PRESS Final October 25, 1989 BLOOD AND BONE MARROW RICHARD D o IRONS, I4.T.p P h o D Molecular Toxicology and Environmental Health Sciences Program School of Pharmacy and Dept. of Pathology School of Medicine -- 2 3 c : Univ. of Colorado Health Sciences Center, C-2.38 L 4200 E. 9th Ave. Denver, Colorado 80262 BLOOD AND BONE MARROW Richard D. Irons Table of Contents Y #1 HD #2 HD #I HD #2 HD #2 HD #2 HD #1 HD #2 HD #2 HD #1 HD #2 HD #2 HD #2 HD #2 HD #1 HD #2 HD #2 HD #2 HD Introduction Blood cells Structure and Cell Biology Gross and microscopic anatomy Hematopoiesis Cells of the blood Xenobiotic Exposure and Metabolism Introduction Metabolism of myelotoxic compounds Mechanisms of Toxicity Introduction Direct toxicity to circulating cells Bone marrow suppression Clonal hemopathies Methodology f o r the Evaluation of Myelotoxicity Introduction Evaluation of toxicity Analysis of stem cell populations #1 HD Introduction #2 HD *I Blood Cells The cells of the blood and bone marrow constitute a widely dispersed and highly regulated organ system, that provides a number of critical functions for the host. Erythrocytes provide a means to transport and maintain hemoglobin for the delivery of oxygen, and act as a buffer to faciljtate the removal of carbon dioxide. Granulocytes serve as a nonspecific, or constitutive, host defense against foreign organisms, functioning both as phagocytes and as mediators of inflammation. Lymphocytes serve as specific mediators of immune response; final arbiters in the recognition of foreign antigens. In addition, certain subpopulations apparently play a role in the regulation of hematopoiesis. Macrophages can be considered together with their circulating precursors, monocytes. They fulfil an intermediate role between constitutive host resistance and immune response, by functioning either as end-stage effector cells in phagocytosis and/or cell killing, and/or as regulatory cells in hematopoiesis and the immune response. Finally, platelets play an important role in hemostasis and in maintaining vascular integrity. When considering target organ toxicity, individual cell lines are often considered separate systems; however, all cell lines share a common origin (the pluripotent stem cell of the bone marrow), are interrelated, and are coordinated during hematopoiesis. Rarely is there a significant adverse effect solely on one cell line. Experimental hematology has witnessed 1ogarithmic growth, and an increase in sophistication over the last few years. This growth has been brought by advances in cell and molecular biology. This chapter will attempt to discuss toxicologic pathology of the blood and bone marrow in the context of our growing, but incomplete, knowledge o f the molecular and cellular events that regulate hematopoiesis. # I HD Structure and Cell Biology #2 HD Gross and Microscopic Anatomy #3 HD Mass and distribution o f hematopoietic tissue In the normal adult, hematopoiesis is largely confined to the medullary cavity of bones. In man, dog and rabbit, hematopoietic tissue occupies all medullary space at birth, but becomes progressively limited to the proximal epiphyses o f the long bones, central skeleton and skull as the individual ages. Even so, adult bone marrow constitutes 1 - 5% of the body mass, depending on the species. In contrast, essentially all medullary space is occupied by functioning hematopoietic tissue throughout 1ife in mice, and rats. Increased hematopoietic activity in adult humans is confined to bone medullary spaces, whereas extramedullary hematopoiesis in the spleen, liver and lymph nodes occurs only under conditions of extreme demand. In fact, hematopoiesis is restricted t o the bone marrow lumen even under the radical conditions of bone marrow transplantation, following total bone marrow destruction by whole body irradiation. In contrast, isolated islets o f erythropoietic cells are normally found in the spleen of the rat, and occasionally small clusters can be found in the liver. In the mouse and rat, the spleen stands poised to become a major hematopoietic organ under the influence of increased demand. The unique predisposition of the spleen t o serve as a hematopoietic organ in the rodent is an important consideration in the use of mice and rats as animal models. Compensatory splenic erythroid, lymphoid, or myeloid hyperplasia may confounding the differential diagnosis of splenic myel oprol i ferative disease, lymphoma or other neoplasia. To add further confusion, most kinetic parameters used to assess the effect of drug, or chemical exposure, on hematopoietic cell prol iferation assume the mass of hematopoietic tissue remains constant. Therefore, the relative number of lymphocytes or macrophages, in addition to their contribution t o the overall prol iferating splenic cell population may vary considerably depending on the degree of splenic extramedullary hematopoiesis encountered. These sources o f variation are particularly important in the assessment of the effect splenic hematopoietic effects in in vitro, as most assays o f immune function in the rodent rely on the spleen as a convenient source of lymphocytes or macrophages. #3 HD Microscopic Anatomy Bone marrow contains reticular cells that form a three dimensional latticework in which hematopoiesis occurs. Anatomically and functionally, it . .. can be divided i n t o vascular and extravascular compartments. Blood enters the marrow via the nutrient a r t e r y , which bifurcates t o form the central a r t e r y of the medullary canal. Radial or medullary a r t e r i e s then branch from the central artery forming a r t e r i o l e s , some of which terminate in c a p i l l a r i e s r w i t h i n the medullary space, while others penetrate the endosteum. These c a p i l l a r i e s supply blood which mixes with blood from the periosteum. Blood returns by means of the vascular sinuses t o the central vein. Hematopoiesis takes place within the convoluted extravascular spaces of the bone marrow sinuses. These sinuses are lined by a single layer of endothelial c e l l s which i n t e r d i g i t a t e w i t h a d v e n t i t i a l , r e t i c u l a r , or d e n d r i t i c c e l l s . Their f i b r e s form the numerous compartments of the extravascular space and support cords o f hematopoietic c e l l s . These stromal c e l l s , which a r e adherent in culture, play a major r o l e in the regulation of hematopoiesis. They form the niches in which hematopoiesis takes place, retract t o faci 1i t a t e transmural migration of blood cell s through the endothelial lumen. Cultured bone marrow stromal c e l l s have been shown t o support hematopoiesis i n v i t r o , and t o produce a variety of growth factors essential f o r the support and regulation o f pro1 i f e r a t i o n and d i f f e r e n t i a t i o n of hematopoietic cells. Trentin introduced the concept o f the "hematopoietic inductive microenvironment" t o describe the influence o f these stromal c e l l s on stem c e l l function and the proliferation of hematopoietic precursor cell s. ~ ~~ T r e n t i n , 1971 Although there is no evidence for any specific organization or localization o f precursor cells within the extravascular cords o f the bone marrow, the distribution of cells within individual niches does not occur at random. ;lusters of differentiating cells of a given lineage are often seen within an individual niche. For example, erythroblasts are often observed forming rosettes around a centrally placed dendritic or macrophage-1i ke cell ; megakaryocytes are frequently found adjacent to bone marrow sinuses, which undoubtedly facilitates the release of platelets directly through the sinus wall. There are no major species differences in the architecture of the bone marrow. As previously mentioned, the entire medullary space o f mice and rats is occupied by hematopoietic tissue throughout 1 ife; therefore, the bone marrow of these species contains little or no fat. In contrast, adult bone marrow in man, dog and rabbit contains many adipose cells. Bone marrow of the rat can be distinguished from mouse and man by larger numbers of mast cells with prominent basophilic granules. In fact, careful preparation of bone marrow smears from the rat is important to minimize the disruption o f mast cells whose granules can obscure other cells. #2 HD Hematopoiesis In the normal steady state, senescent blood cells are continually replaced by cells originating in the bone marrow. This replacement amounts to 2- 3 x 10" o f cells/day for an average human adult. Hematopoiesis i s maintained and regulated through a process of amp1ification and differentiation, in which immature progenitor cells give rise to a larger number of progressively more differentiated generations o f cells (Figure 1). The hematopoietic cells of the bone marrow are divided into four compartments, based of their tendency to differentiate and their prol iferative capacity. At the apex of this pyramid i s the pluripotent stem cell (PSC), which is capable o f giving rise to progenitor cells in any lineage, but appears to have a very limited proliferative capacity even though it has unlimited self-renewal characteristics. With each division in progressive differentiation of the PSC an associated increase in proliferative capacity and a corresponding decrease in self-renewal potential occurs. Lineagecommitted or mortal progenitor cells, have a more limited repertoire of gene expression, but an increased tendency to pro1 iferate enabl ing them to produce precursor cells in 1-3 of the hematopoietic 1 ineages. The morphologically recognizable and highly prol i ferative precursors o f the various cell 1 ineages give rise to the end-stage cells of the blood. With the exception of lymphocytes and monocytes, end-stage blood cells have no prol i ferative capacity. #3 HD Stem and progenitor cells A tribute to the insight o f hematologists early in this century is the observation that all blood cells were descendants o f a common stem cell as early as the 1930's. Not until 30 years later was the clonal nature o f hematopoiesis experimentally confirmed. Pluripotent stem cells (PSC) represent an exceedingly small compartment of cells in bone marrow (1 in lo3 lo5 nucleated cells, depending on the species). Historically, direct studies on the behaviour and regulation of stem cell populations were, and still are, hampered by the lack of reliable morphologic criteria. Despite success in PSC identification and purification, stem and progenitor cell populations continue to be defined functionally by clonal assays, because of their scarcity. #4 HD Y COLONY-FORMING UNIT-SPLEEN (CFU-S) ,.I reconstitution experiments with irradiated mice, Ti I I and McCulloch observed that certain bone marrow cells had the ability to form hematopoietic colonies in the spleens of lethally irradiated syngeneic recipient mice. They observed that this event coincided with the capacity for self renewal and the abi1ity to produce cells of the myeloid, erythroid, and megakaryocyte 1ineages. Since then, the colony-forming unit-spleen (CFU-S) assay has been widely adopted as a quantitative assay for the estimation o f PSC in mouse bone marrow. Subsequent studies demonstrated that a subset o f cells within the CFU-S are capable o f giving rise to all hematopoietic and lymphoid lineages. The CFU-S pool is now recognized to consist of a heterogeneous group of cells, remarkably variable in their individual capacities for proliferation, differentiation and self-renewal. Two subsets o f CFU-S can be distinguished on the basis of their propensity for self-renewal and differentiation: CFU-S I, which have greater capacity for self-renewal and proliferation and are noncycling, and CFU-S I1 which have much less self-renewal and proliferative capacity but cycle actively . New culture techniques for stem cell populations in semisolid media have revealed overlapping cell populations, with characteristics intermediate between PSC and committed stem cell populations. The CFU-Mix cell, for example, is capable of giving rise to myelocytic, erythrocytic and potentially lymphocytic cell lineages, yet apparently has 1 imited capabilities for selfrenewal. At present, it is thought the CFU-S I1 and CFU-Mix are the same cell popul ations detected by different assay procedures. c To date, equivalent assays for CFU-S have only been characterized in mice and rats, although there i s evidence that a similar cell may colonize the spleen in dogs following radiation. However, a number o f in vitro culture systems have been developed that permit the enumeration of committed or progenitor cell lines in a variety of species. #4 HD Progenitor Cells Characterization of early committed progenitor cells followed studies in the mid 1960's which showed colonies of individual progenitor cells could be grown in culture supported by conditioned medium. Subsequently, a variety o f growth factors or colony stimulating factors (CSF's) have been identified that support the growth of specific hematopoietic precursor cells in vitro. The most primitive erythroid progenitor cell is the burst forming unit-erythroid (BFU-E), which gives rise to large colonies of erythroid precursor cells which appear after 6 days in semisolid culture in the presence of high concentrations o f erythropoietin (Epo). BFU-E is now thought to represent a very early cell in the commitment process, that is responsive to IL-3 (see below). The cell directly responsive to Epo is the colony forming uniterythroid (CFU-E) which gives rise to smaller colonies of erythroid cells within 2 - 3 days of culture on semisolid media. - Colonies consisting of predominantly granulocytes, macrophages, or a mixture of both, can also be obtained from a culture of bone marrow cells in semisolid media depending on the source of humoral stimu1 ation used. These cells have been classified as colony forming unit-granulocyte-macrophage (CFU- r GM) and colony forming unit macrophage (CFU- M). Additional colonies have been identified in culture for various lineages including: megakaryocytes (CFU- CUJ -F Meg), fibroblasts (CFu-F) and pre-B lymphocytes (CFU-BL). There is also 1 imited evidence to demonstrate the existence of a pre-T lymphocyte population in bone marrow. #3 HD Regul ati on #4 HD The Microenvironment The microenvironment plays a critical role in maintaining the immortality of the PSC, and in regulating progenitor cell activity. That hematopoiesis could be subject to regulation by soluble factors was recognized at the turn o f the century when serum from anemic rabbits was shown to increase RBC in normal animals. In addition, there is also evidence t o indicate contact with stromal cells is necessary t o support and regulate stem cell differentiation. The dual importance o f stem cell and stromal cell components in the maintenance of normal hematopoiesis is illustrated by genetic anemia in mice. SI/SId and W/W' mice both exhibit macrocytic normochromic anemia; however, the pathogenesis of the anemia differs. W/W' mice possess a congenital stem cell defect that is correctable by transplantation with normal bone marrow cells, whereas transplantation of stromal cells is necessary to correct the anemia in SI/SId mice. In addition to providing a matrix for hematopoietic cells, the stromal fi brobl asts produce soluble products that regulate hematopoiesis. There is evidence macrophages may regulate stromal secretions through IL-1 c production. T-lymphocytes also participate in the regulation of hematopoiesis. Selective removal of T-lymphocytes from erythroid progenitor cell populations reduces BFU-E in vitro. T-cell depleted bone marrow will not cure anemia i n the W/W' mouse. In addition, pre-sensitized T-lymphocytes produce an antigenspecific increase in eosinophil production in vitro. #4 HD Growth Factors The regulation of hematopoiesis involves a complex set of interactions among growth factors and differentiating cells, mediated via surface receptors on target cells. Expression of these receptors i s linked to differentiation of hematopoietic precursor cells. While cell differentiation may be a stochastic process, it appears to be dependent on the availability of various growth factors. A growth factor may act directly on a progenitor cell, or indirectly via interaction with accessory cells to alternatively stimulate or suppress hematopoiesis; hematopoiesis requires a fine balance between stimulatory and inhibitory factors. Although more detailed knowledge of the number, role and control o f growth factors and receptor expression will be forthcoming, a rudimentary understanding o f these factors is available (Table 1 ) . Several growth factors - have been molecularly cloned and purified, and are the subject of intensive laboratory and clinical investigation. Interleukin 3 (IL-3), originally coined "Mu1ti -CSF" and subsequently c renamed, is a product o f T-lymphocytes that serves as a multi-lineage growth or viability factor. It acts at an early stage in hematopoietic cell differentiation, permitting the growth and survival of PSC. In addition, IL-3 supports growth and differentiation of early restricted 1ineage or progenitor cells, such as granulocyte/macrophage, erythroid, megakaryocyte, mast cell and pre-lymphocyte 1 ineages. Two growth factors appear necessary to support the development of erythroid cells: IL-3 and Epo. IL-3 supports the development of PSC and 1 ineage committed progenitor cells including early erythroid progenitors (BFU- E). A complex glycoprotein produced mainly in the kidney, Epo stimulates the development of CFU-E, which appears to be derived from proerythroblasts or their immediate precursors. A variety o f colony stimulating factors (CSF), including macrophage colony stimulating factor (M-CSF) , granulocyte/macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF) are a l l produced by bone marrow fibroblasts and endotheli a1 cells. Monocytes produce M-CSF, and T lymphocytes produce GM- CSF and M-CSF. When studied singly, MCSF, GM- CSF and G- CSF predominantly stimulate the formation of macrophage, macrophage or granulocyte and granulocyte colonies, respectively . - Concentration-dependent interactions between various growth factors are complex. For example, concentrations of GM-CSF stimulate primarily the formation of granulocyte colonies, whereas low concentrations result in formation of macrophage colonies. GM-CSF has also been demonstrated t o P support megakaryocyte and erythroid colony formation. In the presence of GCSF, granulocyte colonies predominate at low concentrations; macrophage colonies are observed only at high concentrations. IL-3 can potentiate the production of progenitor colonies in the presence of various committed growth factors. Synergistic effects following coadministration of IL-3, M-CSF and GM-CSF have been observed in mice both in vivo and in vitro. In contrast to growth factors, a number of inhibitory molecules have effects on hematopoiesis, including: lactoferrin (LF), prostaglandins (PGE) the interferons (XFN) and tumor necrosis factor (TNF). LF is a metal-binding glycoprotein produced by granulocytes which suppresses the re1ease o f GM-CSF and other stimulatory factors. Monocytes and macrophages express surface receptors specific for LF, and some evidence suggests that LF multi-lineage suppression is mediated through suppression of IL-1 production by macrophages. Type E prostaglandins (PGE) exert a complex influence over hematopoiesis by selectively inhibiting macrophage precursor cells and stimulating CFU-GM. PGE, makes cells of multiple lineages responsive to feedback inhibition by the induction of Ia surface antigen expression; it also stimulates the release of Epo by the kidney and stimulates erythroid colony formation in vitro. Interferons are a family of glycoproteins, originally studied for their antiviral activity, that suppress hematopoietic progenitor cells. IFN-gamma has been imp1 icated in the pathogenesis of aplastic anemia and, in conjunction with IFN-alpha, synergistically suppresses CFU-mix, BFU-E and CFU-GM. Tumor Necrosis Factor (TNF), from macrophages and natural killer cells, has a cytolytic effect on certain tumor cells, and stimulates the release of IFN and IL-1. P In addition to stimulation of progenitor cells, many CSF's have effects on mature end-stage cells. For example, G-CSF and GM-CSF are chemotactic for migrating neutrophils, and stimulate phagocytosis. Thus, in response to bacterial infection, not only do CSF's result in the increased production of the responding cells, but they also prime these cells to meet the specific challenge. Thus, there is considerable evidence to suggest that the cells of the hematopoietic and immune systems collaborate in an elaborate network to regulate and direct hematopoiesis. The use of in vitro studies to evaluate the effects of chemical or drug treatment on the elaboration of individual growth factors holds great promise as an approach to elucidate molecular mechanisms of toxicity to the hematopoietic system. Nevertheless, several important questions remain to be answered before the results of individual studies can be interpreted with confidence. Despite the influence o f the microenvironment and individual growth factors on PSC survival or the growth of lineage-specific progenitor cells, PSC differentiation and pro1 iferation are a random processes. Haw microenvironment and growth factors interact with humoral regulation of lineage-specific progenitor cells is not entirely understood at present, nor is our knowledge concerning the effect of individual growth factors on hematopoiesis in vivo complete. For example, do fibroblasts produce - hematopoietic growth factors in v i v o ? If all fibroblasts (e.g. lung, peritoneum, bone marrow etc.) can produce growth factors, what restricts or regulates this function in specific tissues? By now it should be apparent how complex are interactions among CSFs; demonstrating an effect on the c. elaboration of a single growth factor is unlikely to reflect intricacies of the toxic response in vivo. #4 HD Clonal succession The role o f clonal succession in the regulation of hematopoiesis is of considerable consequence in understanding potential mechanisms of leukemogenesis. The theory of clonal succession was proposed a potential protective or regulatory mechanism for hematopoiesis, in which rapidly prol iferating hematopoietic precursor populations possess a restricted capacity for independent prol iferation. For such populations, terminal differentiation is linked to proliferation. The effect of this linkage is to decrease the effect of genetic or nondisjunctional errors in rapidly pro1 iferating precursor cells; therefore, control of PSCs diminishes the opportunity for incorporation of errors into an immortal cell line. The alternative hypothesis states PSC regulation in vivo is entirelv a stochastic process, and multiple PSCs are activated at any one time. Cloning and growth of individual progenitor cells has greatly facilitated elucidation of PSC regulation; however, controversy still exists concerning the mechanisms that govern PSC proliferation and differentiation. How many PSC are actually responsible for populating the hematopoietic system at any one time? The alternatives are many, or very few. It is known that - the entire hematopoietic system can be reestablished by very few, perhaps only a single PSC, capable o f giving rise to multiple lineages of cells, a process demonstrated in vitro. However, the question of stem cell activation has only recently been approached directly in w. r One approach to address this question has utilized retroviral-mediated gene transfer techniques, which permit high efficiency labelling of individual stem cells, thereby providing a direct means to follow the activation of individual stem cells and their progeny. Recent experiments, utili2 ng vector mediated labelling of hematopoietic stem cells in reconstituted mice indicate 1 or 2 stem cell clones can account for the majority o f hematopoietic cells in the body at any one time. Changes occur in the relative contribution of various clones with time. These results suggest there is a sequential utilization of PSCs. They imply an i n vivo mechanism for temporal control of stem cell activation exists. In contrast, by purifying of PSC populations, reconstitution of mice with increasing numbers of PSCs leads to an increased number of cl ones contributing to spleen or thymic colonies. Other researchers have concluded, based on estimates derived from the variances in individual mutational events in mature erythrocyte populations, multiple PSC activation i s the rule. Resolution of this debate is likely to impact heavily on the future design and interpretation of bone marrow toxicity studies. #2 HD #3 HD #4 HD Cells o f the Blood Erythrocytes Morpho1ogy and Function P The mature erythrocyte in mammals i s an anucleate biconcave disc. T h i s shape maximizes the surface area t o volume r a t i o , c r i t i c a l f o r optimal gas exchange, and i s readily deformable as the c e l l s pass through c a p i l l a r i e s and the r e t i c u l a r network o f the spleen. The normal erythrocyte can pass through apertures l e s s than half i t s own diameter. Deviations i n the shape of the erythrocyte are called poikylocytosis. Such deviations reduce efficiency of oxygen transport, and can occur following d i r e c t c e l l membrane damage o r osmotic shock. Poikylocytosis i s also seen i n disorders of erythropoiesis, such as megaloblastic o r h y p o p l a s t i c anemia. Increased erythrocyte r i g i d i t y accompanies poi kylocytosis may contribute t o reduced erythrocyte 1ifespan. The size of the erythrocyte is subject to considerable species variation (diameters range 3 ~m in g o a t s t o 8pm in humans). Abnormal variation in erythrocyte size, termed anisocytosis, i s usually the r e s u l t o f response t o erythrocyte loss or injury. Accelerated r e l e a s e of reticulocytes (immature RBC) in response t o RBC loss will r e s u l t i n an increase in the mean corpuscular volume (MCV) of erythrocytes , o r macrocytosis, because reticulocytes are 1arger t h a n mature RBC. Abnormal i t i es in precursor c e l l maturation associated w i t h drug-induced dysplasias o r megaloblastic anemia will also r e s u l t in an increase in the MCV. These anemias are usually accompanied by megaloblastic changes in precursor c e l l s in bone marrow o r spleen. They can be distinguished from hemolytic anemias by the absence o f reticulocytosis. Several different terminologies have been applied to the identifiable cell populations in erythroid maturation, only two of which will be employed in this chapter. The reader is directed to current hematology texts for a v more thorough discussion of the subject. The first recognizable erythroid precursor cell in the bone marrow is the proerythroblast or rubriblast. In Romanowsky-type stained preparations, the nucleus of the proerythroblast has .pal e staining chromatin network containing one or two nucleo1 i The cytoplasm i s bl ue without granules. The basophilic erythroblast or prorubricyte a1so has pale-staining nuclei, but has darker blue cytoplasm. As the basophilic erythroblast matures the chromatin o f the nucleus (rubricyte) becomes progressively more condensed. The appearance of a purple or "polychromatic" hue to the cytoplasm marks the transition to active hemoglobin synthesis in polychromatophil ic erythroblasts (rubricytes). The nucleus in these cells further condenses becoming pyknotic, at which stage the orthochromoblast or metarubricyte (nucleated erythrocyte) is formed. Extrusion of the nucleus gives rise to the immature erythrocyte or reticulocyte, which still contains ribosomal subunits that maintain active hemoglobin synthesis for approximately 2 days after release into the circulation. Erythropoiesis i s one of the most impressive examples of differentiation in nature. #4 HD Production and Fate. The 1 ifespan of the circulating erythrocyte varies considerably from one species to another. Unlike granulocytes and lymphocytes, the erythrocyte spends its entire lifespan confined to the circulation. The average erythrocyte lifespan in humans is approximately 120 days compared to about 30 & days in the mouse. The spleen plays a major role i n the sequestration and removal o f damaged RBC, although secondarily, other r e t i c u l a r t i s s u e s f u l f i l t h i s function. Daily turnover of over 10" RBC occurs i n man compared t o lo9 RBC in the mouse. *. #3 HD #4 HD Granulocytes Morpho1ogy The granulocytic s e r i e s consists of three c e l l l i n e s t h a t share common developmental and morpho1ogic features. Mature granul ocytes a r e c e l l s of 10- 20 l m diameter characterized by a segmented nucleus are termed "polymorphonuclear" leukocytes (PMNs). They contain azurophil i c and s p e c i f i c cytoplasmic granules, distinguishable by 1ight microscopy. Classification o f granulocytes into neutrophils, eosinophils and basophils i s based on the staining c h a r a c t e r i s t i c s o f t h e i r primary cytoplasmic granules i n Romanowskystained smear preparations. Azurophilic granules are found in a l l three granulocytic c e l l types. In mammals, the predominant granulocyte i s the neutrophil which has 3-7 lobed nucleus and specific granules that usually stain a beige color. A species difference can be seen in the heterophil o r "pseudoeosinophil" of the rabbit and guinea pig, where the specific granules s t a i n an intense orange p i n k t o red. Eosinophils usually contain 2-3 lobed nuclei and red s p e c i f i c granules, which vary i n size and shape among species. The s p e c i f i c granules o f basophils s t a i n intensely basophilic and vary dramatically in s i z e from one species t o another. - A1 1 three granulocytic cell types exhibit comparable stages in development and differentiation. The first recognizable precursor o f the granulocyte lineage i s the myeloblast. Its nucleus has a delicate pale staining chromatin network, and usually contains three or more large nucleoli. r Myeloblast cytoplasm is pale blue containing a few azurophilic granules, the distinguishing feature of this cell. The increase in the number of azurophil ic granules in the cytoplasm characterizes the promyelocyte. The nucleus o f the promyelocyte is frequently kidney shaped and may contain remnants o f the once prominent nucleoli. Its cytoplasm varies from light blue to the pale pink of the mature granulocyte. As the promyelocyte matures, the chromatin o f the nucleus condenses and a second type o f granule, the specific granule,appears. An increase in the number o f specific granules, together with the presence of a pale pink cytoplasm, are the hallmarks of the myelocyte. As the myelocyte continues to differentiate, the nucleus further condenses to form a rod or "band" appearance. Further nuclear condensation results in the mu1 ti-lobul ated appearance of the mature granulocyte. Nuclei of granulocytic progenitor cells and mature granulocytes in some species often contains a centrally placed vacuo1 e. These "donut" cell s are normally found in rodent bone marrow, and become more prevalent upon stimulation o f granulopoiesis. In humans, their presence is associated with severe pathology, such as that encountered in myelodysplastic syndrome or leukemia. #4 HD Function The cytoplasmic granules o f granulocytes are lysosomes which concentrate a variety o f enzymes and biologically active substances used in phagocytosis, killing and inflammation. The contents of azurophilic granules include: myeloperoxidase, acid phosphatase, esterase, beta-glucuronidase and lysosyme. Neutrophi 1 ic specific granules contain a1 kal ine phosphatase lysosyme and lactoferrin. Eosinophilic granules are rich in plasminogen as well as a c variety of hydrolytic enzymes including: acid phosphatase, glucuronidase, phospholipase, cathepsins and ribonuclease. A1 though eosinophil ic granules exhibit peroxidase activity, this activity is different to myeloperoxidase of neutrophils. Basophilic granules contain, in addition to a variety of enzymes, heparin and histamine. The primary function of granulocytes is bacterial phagocytosis and .ki 1 1 i ng Secondary functi ons i ncl ude the re1 ease of pyrogens and 1 actoferri n , the latter serving in a feedback loop to control granulopoiesis. Certain cellular functions are critical to the role of granulocytes in the circulation. Among these are margination or adherence to endothelial cells; chemotaxis, i .e., diapedesis through the vessel wall and migration toward `a chemotactic stimulus; attachment and phagocytosis of bacteria; fusion of lysosomes with phagocytic vacuoles, subsequent degranulation and kill inq. Adherence involves the secretion of lactoferrin and a complement receptor. It is enhanced by complement activation or leukotriene 84 (LT84). Chemotaxis is stimulated by a variety of substances including bacterial products such as endotoxin, activated complement and LTB4. Chemotaxis is dependent on signal transduction through the plasma membrane (including calcium flux), and cytoskeletal integrity. Phagocytosis requires recognition of, and attachment to, a foreign particle. This process is facilitated by opsonization, where the foreign particle is coated by immunoglobulins. Following engulfment o f the particle, the internalized vacuole undergoes fusion with a lysosome. Lysosomal degranulation leads to the activation o f hydrolytic enzymes, and the induction of the leukocyte oxidative burst, which mediate bacterial killing. c In addition to these generalized functions of PMN's, eosinophils are attracted by mediators of allergy, such as histamine and serotonin. Basophils are the circulating equivalent of tissue mast cells, and play a role in immediate hypersensitivity and inflammation. In species such as the rat, which have a relatively high number o f mast cells in the bone marrow and reticular tissues, circulating basophils are exceedingly rare. #4 HD Production and Fate Unlike the erythrocyte, which spends its entire 1 ifespan confined within the circulatory system, the time the granulocyte spends in the circulation is extremely short, i.e., about 10 - 14 hr. Granulocytes leave the blood vessels by passing between endothelial cells in a process known as diapedesis. After diapedesis, the lifespan of the granulocyte in peripheral tissues is thought to be 2-3 days. A major route of elimination of granulocytes involves transmural migration through the gut wall. Because transit time is short, and lifespan in peripheral tissues o f the granulocytes i s only days, decreased production or increased destruction of granulocytes rapidly effects the circulating pool. As a result, granulocytopenia is frequently an early finding in bone marrow suppression. [Insert Figure 2 here] The granulocyte pool is unevenly distributed between the maturation and storage compartment of the bone marrow (90%) and the circulation (10%) (Figure 2 ) . Granulocytes in the peripheral blood are equil i brated between two r compartments: circulating and marginated. Circulating granulocytes comprise that population accessible during routine blood counts. An equal number of cells are marginated, or adhered to the endothelial wall, and can rapidly released into the circulating pool. Since the peripheral blood granulocytes (both circulating and marginated) constitute only about 10% of the total granulocyte maturation/storage pool, the release of only a very small proportion of storage/maturation compartment can result in a doubling of the peripheral granulocyte count. G1ucocorticoids, f o r example, produce granulocytosis, due to release o f cells from the bone marrow compartment, decreased adherence and decreased egress. #3 HD #4 HD Monocyte-Macrophages Morpho1ogy Macrophages comprise an array of tissue-specific and circulating cells derived from bone marrow precursor cells and the circulating monocyte. These cells include: alveolar macrophages, the Kupffer cells o f the liver, tissue histiocytes, splenic, lymph nodes, and bone marrow macrophages. The typical circulating monocyte is a large cell, about 30 microns in diameter, containing a large irregularly shaped nucleus. In Wright's or Giemsa-stained preparations, the nuclear chromatin is del icately filamentous. Its abundant cytoplasm, is sky blue or grey and may contain numerous fine azurophilic granules and vacuo1es. A1though a bl ast cell with characteristics resembl i ng monocytes is frequently observed in bone marrow preparations and is defined as a "monoblast", a morphologically identifiable maturation sequence of monocyte precursors in the bone marrow has not been identified. #4 HD F Function Macrophages are phagocytes, and function to remove foreign bodies, parasites and bacteria; however, significant tissue specific differences in individual macrophage phagocytic functions exist. For example, intravenous carbon particles given to rats are more efficiently removed by Kupffer cells than by splenic macrophages, whereas the opposite is true for foreign or damaged RBC. The fate of administered antigens varies markedly from one species to another. Intravenously administered particulate antigens accumulate primarily in the liver of rats, whereas the lung i s the primary site for deposition of blood borne particulates in sheep and goats. Mice and rats clear inhaled particulates much more rapidly from the lung than dogs. Differences in the fate of antigens and their pattern of distribution may play a significant role in explaining major species differences in immune response and disease patterns. Bone marrow macrophages are now thought to play a supportive role in hematopoiesis through the secretion of IL-1 which in turn stimulates the re1ease of CSF's from fibrobl asts, T-lymphocytes and endothel i a1 cells. Finally, macrophages play a major role in antigen presentation and regulation of immune response. #4 HD Production and Fate Monocytes have an intravascular l i f e s p a n of 1-3 days. They leave the blood stream a t random. There does not appear t o be any s i g n i f i c a n t return t r a f f i c once monocytes have l e f t the circulation. Although the transformation P o f monocytes t o macrophages has been long a p p r e c i a t e d , whether these c e l l s can normally undergo further p r o l i f e r a t i o n a f t e r leaving the bone marrow is s t i l l under debate. The l i f e s p a n of macrophages i n tissues i s on t h e o r d e r of months. #3 HD #4 HD P1 ate1ets Morpho1 ogy Platelets a r e granular cytoplasmic fragments t h a t take on a v a r i e t y o f shapes depending on their s t a t e of a c t i v a t i o n and the q u a l i t y of the smear. Their size i s a l s o v a r i a b l e , depending on the preparation and the s p e c i e s . They have a marked tendency t o a g g l u t i n a t e and o f t e n form l a r g e a g g r e g a t e s on p e r i p h e r a l blood smears. P l a t e l e t s a r e g e n e r a l l y round, oval or t e a r d r o p i n shape, measuring between 1 and 5 microns i n diameter. Despite their amorphous appearance, pl atel ets are highly organized structures. The surface o f the p l a t e l e t i s rich i n g l y c o p r o t e i n s and a c i d mucopolysaccharides. Their cytopl asm c o n t a i n s numerous m i c r o f i l a m e n t s , i s supported by a network o f microtubules, and is s l i g h t l y b a s o p h i l i c c o n t a i n i n g purple granules. Platelet granules contain a variety of factors that accelerate c l o t t i n g , promote c l o t contraction, and growth f a c t o r s which promote wound h e a l i n g . These i n c l u d e : S e r o t o n i n , e p i n e p h r i n e , f i b r i n o g e n and platelet derived growth factor. Enzymes of the cyclooxygenase pathway are also present for the synthesis of prostaglandins and thromboxanes. Megakaryoblasts are the earl i est recognizable precursor cells of the c thrombocytic lineage, and are extremely large cells, measuring up to 100 rm in diameter. They are distinguished from other hematopoietic precursor cells in that they are polyploid. They undergo a sequential maturation to megakaryocytes, involving nuclear segmentation and the acquisition of cytoplasmic granules before fragmenting to form platelets. During platelet formation, megakaryocytes extend filamentous cytoplasmic projections through the endothelial wall which break off releasing platelets directly i n the circul ati on. #4 HD Functi on Platelets play a major role in hemostasis through the initiation of clot formation. They are activated by contact with collagen, air or foreign surfaces. Activation (usually via adhesion) brings about the release of mu1 tipl e factors causing aggregation, increased adherence to endotheli a1 surfaces and the release of clotting factors I 1 1 and IV. Platelets also release growth factors, such as pl atelet-derived growth factor (PDGF), thought to promote tissue regeneration following injury. #1 HD Xenobiotic Exposure and Metabolism Hematotoxic compounds can be divided into two major categories: r pharmaceuticals and occupational hazards. Exposure routes used in metabolic and toxicity studies include parenteral, oral or inhalation routes. The influence of xenobiotic biotransformation and pharmacokinetics on bone marrow toxicity is a complex; many compounds that produce bone marrow toxicity require bioactivation, but exceptions exist. Major exceptions appear to be reactive antineoplastic agents, such as hydroxyurea, 5-azacytidine, the nitrogen mustards, and antibiotics such as puromycin. For many myelotoxic agents, metabolic studies are complicated by their chemical reactivity. For example, studies on the pharmacokinetics and fate of the nitrosoureas have been hampered by their rapid degradation. Such difficulties are often further compounded by unequal distribution indifferent tissues, a fact highlighted by studies on the nitrosoureas and most recently 1,3-butadiene. Furthermore, detailed comparative studies of species differences in xenobiotic metabolism, pharmacokinetics and disposition, have rarely been performed simultaneously with experiments designed to measure bone marrow toxicity. As a result, it is often difficult to interpret the precise role of secondary and tertiary metabol ism in myelotoxicity. The reactions associated with the metabolism of hematotoxic agents are the same as for other xenobiotics; they involve primarily oxidation, hydroxyl ation, demethylation, deamination, conjugation and nitroreduction. Not surprisingly, the liver i s the principal site of primary oxidative metabolism; however, in certain situations there is evidence that bone marrow metabolism plays a major role in conferring susceptibility. For example, there is evidence that tertiary metabol ism of the hydroxy-metabol i tes of benzene in the bone marrow play a major role in target organ toxicity 2. Y Although increasing attention is being given to the role of bone marrow in the bioactivation of foreign compounds and metabolites, the role if any o f tissue specific detoxification in hematotoxicity is unknown. #2 HD Metabolism of Hematotoxic Compounds Cyclophosphamide is a classical example o f a compound that has no intrinsic cytotoxicity but requires bioactivation via the cytochrome P-450 mixed function oxidase system to produce the alkylating secondary metabolites, phosphoamide mustard and acrolein. 4-hydroxycyclophosphamide, the primary metabolite, is not as biologically active. Benzene also requires metabolism in order to produce bone marrow toxicity; however, the pattern o f bioactivation and detoxification is complex, as both induction as well as inhibition o f benzene metabolism protect against myelotoxicity. This contradiction can be best explained by elimination and detoxification. Benzene is volatile and rapidly eliminated via the lungs in the absence of metabolism. Induction o f nonspecific biotransforming enzymes results in increased metabol ite-conjugate formation and increased elimination. Benzene bone marrow toxicity correlates with target organ concentrations of the secondary metabol i tes, hydroquinone and catechol, not the primary * Eastmond et a.,1987 metabolite phenol. The particular susceptibility of the bone marrow to benzene toxicity may relate in part to the high level of myeloperoxidase activity in that tissue. Phenol, catechol and hydroquinone are excellent substrates for peroxidase-mediated reactions, giving rise to SH-reactive c benzoquinone metabol i tes. Admini stration of phenol and hydroquinone exacerbates toxicity, suggesting phenol -induced stimulation of hydroquinone metabolism and benzoquinone formation. More recently, the formation of transmuconaldehyde has a1so been suggested as the toxicant responsible for benzene toxicity, as it has reactive properties very similar to the quinone metabolites o f benzene. Despite marked differences in experimental design or routes o f exposure, chronic target organ toxicity is restricted to peroxidaserich tissues such as bone marrow, Zymbal, Harderian and mammary glands. Nitroreduction has been imp1icated in the bioactivation of several nitroaromatic compounds including nitrobenzene and dinitrotoluene. The production of methemoglobinemia by these agents is largely dependent on nitroreduction by bacteria in the gut, a process required for the bioactivation o f chloramphenicol. Chloramphenicol causes a dose-dependent myelosuppression; however, the re1ationship between this reversible phenomenon and the occasional "idiosyncratic" chloramphenicol-induced irreversible aplastic anemia is not clear. Nitroso-chloramphenicol produces an irreversible inhibition of CFU-S in mice in vivo and CFU-C i n human bone marrow cells in vitro, indicating this compound is the most likely toxicant. A tendency for hematotoxic compounds to give rise to very stable metabolites of intermediate reactivity, allowing transport to tissues distant from the site o f primary metabolism (i.e. from the liver to the bone marrow), - is a constant finding in hematotoxicity. This observation has most recently been illustrated by the potent murine leukemogen, 1,4-butadiene for which the bone marrow is a major target organ. Although primary metabolism of the compound occurs predominantly in the liver, the major metabolites, c monoepoxybutene and diepoxibutane, possess long half 1 ives relative to most biologically active aromatic and a1 iphatic epoxides. #1 HD Mechanisms o f Toxicity Hematotoxicity is manifested by altered circulating mature blood cells, both in number and/or interference with their function. A reduction in the number of circulating blood cells results either from excessive destruction (e.g. acquired hemolytic anemia) or suppression o f production. These lesions are classified as granulocytopenia, thrombocytopenia, lymphocytopenia or pancytopenia, depending on the cell type predominantly affected. Anemia is a decrease in the number o f circulating RBC, a decrease in their corpuscular volume (MCV), a reduction in hemoglobin content (MCH) or any combination thereof. Anemias can result from increased destruction of erythrocytes, decreased production of erythrocytes, or both, and are usual ly classified according to the predominant pathology. In making a differential diagnosis o f anemia it is important to consider causes of anemia, other than xenobiotic induced, eg. acute blood loss, nutritional deficiencies (eg. 4 J - b S?kC\T ironrvi tamin B12 or folate) and congenital abnormalities of the erythrocyte membrane hemoglobin synthesis or metabolism. Certain xenobiotics can act as metabolic antagonists, producing anemias in some species similar to those encountered in nutritional deficiencies or pernicious anemia in man (eg. - aminopterin induced megaloblastic anemia). Finally, anemia is frequently an early secondary finding in 1eukemi as and myel odysplasias. -. Decreases in one or more of the leukocyte series or platelets are 9 classified as granulocytopenia, lymphocytopenia or thrombocytopenia, respectively . Because of complex transi t kineti cs and the smal1 proportion of granulocytes and lymphocytes in the circulation relative to storage compartments, transient fluctuations in the numbers of circulating cells are common. Distinguishing between transient cytopenias resulting from non- specific toxicity and those occurring as a result of specific toxicity to the blood and bone marrow is difficult. Abnormal increases in circulating cells can vary in severity from a transient granulocytosis resulting from demargination to acute leukemia. Depending on the nature, extent, and chronicity of the injury, bone marrow toxicity may range from a transient suppression of one or more cell type with rapid recovery following cessation of exposure, to myelodysplasia in which blood dyscrasias and aberrant stem cell regulation may persist or progress to aplastic anemia or leukemia. #2 HD #3 HD Direct toxicity to circulating cells Hemolytic Anemia Acquired hemolytic anemia is among the most frequently reported drugreaction in man, but i s usually of minor severity. Decreased lifespan of circulating erythrocytes defines hemolytic anemias, and can range from subtle RBC membrane changes resulting in premature removal by phagocytic cells, to - rupture with a subsequent life threatening hemolytic crisis. It is common to see a minor hemolytic component in bone marrow suppression type anemias. #4 HD 7 D iagnosis Laboratory findings in hemolytic anemia vary according to the severity and cause of the disease. Plasma or serum hemoglobin concentration are still the most sensitive indicators in routine use; however, decreased erythrocyte survival time, as measured by non-routine isotopic label1ing techniques, may be the only finding. Although increased in unconjugated bilirubin may be seen following increased hemoglobin catabol i sm, it is neither a very sensit ve or specific indicator of hemolytic anemia. If the rate of hemolysis is significant, a decrease in RBC and Hct (or PCV) may be observed. Hemo ytic anemias are often accompanied by a regenerative response manifested by hyperplasia of the bone marrow and increased circulati ng reticulocytes, which may increase the MCV as reticulocytes are larger than mature RBC. The peripheral blood smear may show morphologic evidence of erythrocyte destruction including erythrocyte fragments (schizocytes), cells exhibiting 1oss of membrane (spherocytes), RBC agglutination, or Heinz body formati on, depending on the pathogenesis of the anemia. Because schizocytes and spherocytes have abnormally low cell volumes, the MCV may not be abnormal as these small cells are balanced by reticulocytes. The coefficient of variation in RBC size may increase dramatically. Splenomegaly i s frequently encountered in chronic hemolytic anemias in rodents because of sequestration of damaged erythrocytes and the presence of - extramedullary hematopoiesis. C1usters of erythroid precursor cells may be found in the red pulp along with hemosiderin-laden macrophages; the entire red pulp may be occupied with erythroid precursors - care must be taken not to confuse the histologic picture with that of lymphoma. Although the presence C of basophilic and polychromatophilic erythroblasts can usually be discerned in routine hematoxyl in-eosin stained sections, Wright-Giemsa stained sections or imprints are useful for cell identification. Heinz bodies, intracorpuscular precipitates of denatured hemoglobin, indicate oxidative damage to hemoglobin. They constitute the major cause of erythrocyte destruction in hemolytic anemias associated with glucose-6-phosphate dehydrogenase deficiency. A number of attempts have been made to characterize animal models of hemolytic anemia associated with genetic deficiencies, but many have proved disappointing as experimental surrogates for man. For example, sheep have been characterized with 10% of normal human G-6-PD, but they are insensitive to drugs normally associated with hemolytic anemia in G-6-PD deficient humans. #4 HD Mechanisms o f chemically induced hemolytic anemia A number of agents are associated with secondary hemolytic anemia in man. Agents are classified as immune mediated, oxidative stress (exposure to potent oxidizing agents, or through increased susceptibility due to congenital glucose-6-phosphate dehydrogenase deficiencies), and non-oxidative. Several excellent detailed reviews are already avail able on hemoglobinopathies and hemolytic anemias secondary to drug or chemical exposure in man and will not be repeated here - #3 HD #4 HD c Cytopenias/Cytoses Granulocytopeni a. A number of xenobiotics that alter granulocyte adherence are known to markedly alter the equilibrium between the circulating and marginated compartments. Ethanol, colchicine and epinephrine can rapidly increase the circulating granulocyte pool via demargination, but the resulting granulocytosis does not reflect an increase in host resistance as chemotaxis, phagocytosis and ki 1 1 i ng are suppressed. Histamine, iron oxide and dextran produce an apparent granulocytopenia by increasing the size o f the marginated pool. #4 HD Thrombocytopenia. Thrombocytopenia occurs following increased destruction, consumption, aggregation or sequestration o f platelets. Large cavernous hemangiomas, massive hepatic necrosis, and hypersplenisn can elicit thrombocytopenia via platelet sequestration and consumption. Hypersplenism induced by methylcellulose injection in rats results in thrombocytopenia which can be corrected following splenectomy. Beutler, 1985; Smith, 1986. Direct affects on platelet surv Val are immunologic or non-immunologic, depending on whether antibody complex formation is involved. The precise role of complement-mediated mechanisms of mmunologically induced cytopenias is not clear; however, platelets can adsorb complement fixing antibodies by a variety P o f mechanisms. Xenobiotics can bind to platelets and act as haptens or promote the binding of immune-complexes to platelets by other means. Antibody complex binding can trigger direct complement-mediated platelet lysis or stimulate serotonin release. Alternatively, platelets binding C, and Ig are sequestered and phagocytosed. Agents thought to act directly on platelets include the antibiotic, ristocetin, which induces agglutination, and heparin which produces a transient thrombocytopenia, though a poorly understood mechanism. #2 HD #3 HD Bone marrow suppression Cell cycle Kinetics Bone marrow suppression is the most frequent side effect of cancer chemotherapy. Rapidly pro1 iferating cells demonstrate unique susceptibility t o certain cytotoxic drugs compared to their resting counterparts, an effect attributed t o their relative position in the cell replicative cycle. The cell cycle can be divided into four phases relative to DNA synthesis: M, mitosis, (physical cell division); GI, the first gap or period of no measurable DNA synthesis; S, the phase o f active DNA synthesis; G,, the second gap after DNA synthesis is complete, in which the cell contains twice the diploid content of DNA found in G,. Cells not in active cycle containing the normal diploid content of DNA reside in the Go phase (Figure 3 ) . G, can be further divided into subcompartments relative to quantitative and qualitative differences in RNA and protein synthesis. [Please insert Figure 3 & 4 here] b. The committed stem cell populations of the bone marrow exhibit a pattern of asynchronous exponential growth, in which cycling cells move through the cell cycle independent of one another, so the probability that a cycling cell will be in any particular phase of the cycle at a given point in time i s proportional to the amount o f the replication cycle that is spent in that phase. The proportion o f asynchronously dividing cells in any given phase can be depicted by a histogram based on the DNA content of cells (Figure 4 ) . Although many agents are selectively toxic to rapidly dividing cell populations, there can be considerable variation in the response o f proliferating precursors, progenitor and stem cell populations to toxicity. Some agents show specificity for stem cell populations which favour selfrenewal. Potent alkylating agents, such as busulfan or the nitrosureas, target resting PSC in addition t o other proliferating cells, thereby suppressing PSC function and number. Nitrosureas produce delayed and severe bone marrow damage, 1- 2 months after admin stration. Similarly, residual stem cell injury occurs long after cessation o f treatment with busulfan. Stem cell populations in S phase are more resistant to radiation injury than are resting cells, whereas cyclophosphamide preferentially affects stem cells with low self-renewal potential and high proliferative activity. Other alkylating agents, such as actinomycin D are similarly classified as cycle- specific agents; they selectively target cycling cells in any phase. Bichloro-ethyl-amines, mephalan, chlorambucil and nitrogen mustard, are examples of compounds that are also cycle-specific. These agents typically produce a dose-dependent bone marrow suppression that i s often followed by r protracted recovery. Some compounds such as colchicine, the vinca alkaloids, and methotrexate, target cells in a specific phase of the cell cycle, and produce a plateau-type of dose response curve. Colchicine and the vinca alkaloids interfere with microtubule assembly and mitotic spindle formation, which results in an arrest of cycling cells in G,/M. Susceptibility is confined t o cells in late S and early G, phase at the time of exposure. Since a proportion of asynchronously dividing cells are in this part of the cycle at any given time, increasing the dose of a phase-specific cytotoxic agent beyond an effective threshold does not increase the number o f affected cells (Figure 4) For cycle-or phase-specific agents, the frequency and duration o f exposure can be an important factor in determining toxicity, because altering the timing of exposure relative to cell cycle (eg. varying the interval between doses) can lead to synchronization of the cell cycle, with subsequent alteration of the number of cells susceptible to toxicity at any one time. For example, a single therapeutic dose of cyclophosphamide can result in a transient depression in circulating leukocytes, whereas multiple dosing at two week intervals may result in severe leukopenia, aplastic anemia and death. Benzene appears to share characteristics in common with both cyclespecific and phase-specific agents. It causes an arrest of cycling cells in G,/M. This arrest is seen as an initial increase, followed shortly by a decrease in bone marrow cell turnover, suggesting a defect in the maturation r of precursor cells. Hydroquinone and its terminal oxidation product, pbenzoquinone, have been imp1 icated as the benzene metabolites responsible for the effects of benzene on pro1 i ferating cells. These compounds arrest cycl ing cells in G,/M by inhibiting microtubule assembly, thereby interfering with sulfhydryl-dependent GTP binding t o the tubulin dimer. The effects of benzene are not entirely restricted t o cel s in G, or M phases of the cell cycle, however. At non-cytotoxic concentrat ons, the quinone metabolites o f benzene can inhibit the entry of resting cells into cycle, thus modulating the dose response relationship between responding cells and proliferative stimuli. Direct effects on the cytoskeleton and/or interference have been hypothesized to explain these effects. Benzene produces a marked 1ymphocytopenia and suppression o f progenitor B-lymphocytes in mice. In addition, hydroquinone reduces the ability of stromal cultures t o support granulopoiesis, apparently because of a selective suppression of IL-1 release by macrophages, which results in a reduction in IL-1 dependent IL-4 release by fibroblasts and subsequent pre-B cell suppression. Nitrosourea-induced bone marrow toxicity has been reproduced in a wide variety of animal models; however, relative differences in species susceptibility to these compounds exist. Mice and cats appear to be - particularly susceptible to the effects of nitrosourea compounds on bone marrow, while rats, guinea pigs and sheep appear to be somewhat resistant. A definitive mechanism of nitrosourea-induced bone marrow toxicity has not been established, but direct alkylation or carbamylation o f DNA and RNA have been t. imp1icated as potential mechanisms. In structure activity studies between the chloroethylnitrosureas, which produce significant bone marrow toxicity at therapeutic doses, and chlorozotocin, which does not, carbamylation and the regional pattern of alkylation of transcriptionally active chromatin, have been found to correlate with differences in bone marrow toxicity. #3 HD Megalob1 astic Anemia Erythrocytes are less susceptible than leukocytes to numerical alterations, resulting from interference to hematopoiesis, as they have a long lifespan relative to leukocytes. Therefore, frank anemia is usually a late manifestation of bone marrow suppression, even though erythropoiesis se may be more sensitive to chemical toxicity than granulopoiesis. Bone marrow suppression re1ated anemias rarely occur in the absence of accompanying cytopenia(s) and maturational or "megaloblastic" changes in circulating blood cells. Xenobiotics that interfere with RNA or DNA synthesis, or produce disturbances in cell division frequently produce a megaloblastic anemia. Such agents include folate antagonists (methotrexate), inhibitors of purine o r pyrimidine synthesis (6-mercaptopurine and 5-fluorouracil) and pentose sugar analogues such as cytosine arabinoside. #4 HD Diagnosis Macrocytic and microcytic erythrocytes can be present in megaloblastic anemias that arise from bone marrow suppression. Distinctive morphologic P changes can be seen in precursor cells o f erythroid, granulocytic and megakaryocytic lineages in bone marrow; increase in the number of immature progenitor cells is also present. Nuclei may be abnormally large and possess a delicate chromatin pattern, even in cells with relatively mature cytoplasmic features. Precursor cells in a1 1 1 i neages, and circulating erythrocytes, may exhibit increased numbers o f micronuclei or Howell-Jolly bodies, a reliable indicator of dyserythropoiesis. These finding are particularly evident in the mouse, as micronuclei-containing cells are not effectively removed by the spleen. The frequency o f circulating reticulocytes may be normal or reduced. Circul ating hypersegmented granulocytes may be seen, although the number o f granulocytes or platelets may not be significantly reduced. #3 HD Cytopeni as Reductions in the number of circulati ng pl ate1ets, granulocytes or lymphocytes are the most common manifestations o f bone marrow suppression. However, results may vary, depending on the experimental conditions, the strain or species used, and even the individual animal. Thrornbocytopenias may result from destruction, decreased production or release o f platelets, and are the most frequent blood dyscrasias reported secondary to xenobiotic exposure. A wide variety of drugs have been imp1 icated in immune-mediated thrombocytopenia in man, including quinine and -. heparin, but animal models are not well defined. Thrombocytopenia, with or without leukopenia, is often encountered following exposure to myelosuppressive agents, although the mechanisms remain largely unknown. Species differences exist as to the outcome of toxicity. Cytosine arabinoside r and busulfan, for example, are extremely effective in inducing experimental thrombocytopenia in a wide variety of species. On the other hand, cyclophosphamide administration to BDFl mice has been reported to produce a marked depression in the number of bone marrow megakaryocytes and CFJ-M with only a slight decrease in blood platelets. In contrast, administration of a single low dose of vincristine to BALB/c mice has been reported to result in thrombocytosis without prior thrombocytopenia, whereas high doses o f vinblastine, but not bleomycin, produce a transient thrombocytopenia in rats. The dog appears to be sensitive to estrogen-induced thrombocytopenia, presumably mediated through decreased megakaryocytopoiesis , a1though an immune-mediated mechanism has been suggested for diethylsti1besterol induced thrombocytopenia in this species. Granulocytopenia may be a useful indicator o f myelotoxicity; however, fluctuations in granulocyte numbers frequently occur in experimental animals independent of drug-induced bone marrow suppression. Non-speci fic stress, or systemic chemical toxicity can transiently alter cell cycle kinetics in proliferating bone marrow populations, producing wide fluctuations in circulating granulocyte numbers. Rodents and rabbits are especially susceptible to stress-induced leukocytopenias. These have been attributed to relative species sensitivity to corticosteroid release, although the precise mechanisms are not understood. For this reason care should be taken in the interpretation of acute or transient fluctuations in leukocyte numbers observed within a few days of treatment. Dose dependent responses are more important. For example, lymphocytopenia appears to be the most sensitive and reliable indicator of repeated benzene exposure in a variety o f species. Surprisingly, granulocytes are relatively resistant to benzene, and c granulocytopenia is a late manifestation o f benzene toxicity. #3 HD Aplastic anemia Aplastic or non-regenerative anemia is a syndrome associated with bone marrow failure, characterized by anemia, pancytopenia, and varying degrees of bone marrow hypocellularity. I t is distinguished from other diseases producing pancytopenia, such as leukemia, infiltrating malignancies, or myeloproliferative disease, by histologic examination o f the bone marrow. Histologically, hematopoietic precursor cells may be almost totally replaced by fat, 1eaving a few mature hematopoietic cells interspersed throughout (Figure 5 ) . [Please insert Figure 5 here] Aplastic anemia is classified as idiopathic or secondary depending on whether i t s onset can be attributed to known causes, eg. radiation, drug or chemical exposure. The syndrome carries a grave prognosis, as in the absence o f successful bone marrow transplantat on, approximately 40% o f all human cases die within 6 months o f diagnosis Aplastic anemias arising secondarily t o chemotherapy or xenobiotic exposure carry an even more dismal prognosis. Aplastic anemia is a disorder of stem cell regulation. Differing theories on the pathogenesis of the disease emphasize the respective roles of hematopoietic stem cells and stromal cells in its evolution. Aplastic anemia has been suggested as a disorder o f PSC which, either through exhaustion o f II numbers or a defect in differentiation, are unable to recapitulate blood cells. There is also evidence that stromal cell defects may play an important role in chronic bone marrow failure, but the success of HLA-matched allogenic marrow transplants in man, and the inability of growth factors to ameliorate the disease, suggests that microenvironmental defects alone cannot completely explain aplastic anemia. In many cases of aplastic anemia, the hypoplastic marrow i s devoid of hematopoietic precursor cells; however, residual islets of erythroid or granulocytic precursor cells may persist. In some of these cases there is evidence to support a clonal origin for aplastic anemia. Animal models o f aplastic anemia are relatively few, and have been largely restricted to those induced by viruses, busulfan, irradiation or benzene. A problem frequently encountered is animals die due to anemia, hemorrhage o r infection, before fulfilling the stringent criteria for a diagnosis of aplastic anemia. A rapidly developing neutropenia, thrombocytopenia and anemia accompanied by a decrease in bone marrow cellularity of CBA mice has been described following infection with Rauscher leukemia virus. Morphological examination o f bone marrow from these animals revealed a maturation arrest in the granulocyte series. It was concluded that the underlying mechanism involved a disorder in differentiation, probably at the level o f a common precursor cell. Busulfan-induced aplastic anemia i s the best studied model o f chemically-induced bone marrow failure. In Swiss mice given an initial treatment of four doses, busulfan produced a transient bone marrow hypoplasia followed by a normal blood picture; by 240 days 80% of the mice died with r pancytopenia. In some instances, no differences in bone marrow cellularity or peripheral blood counts were noted during the latent phase, although lymphocytopenia was a common finding. Subsequent studies revealed that CFU-S and granulocyte progenitor cells were decreased fol1owing busulfan treatment, and were further suppressed following frank bone marrow failure. In reconstitution studies, bone marrow cells from busulfan treated mice grew poorly when inoculated into irradiated mice, providing additional support for a stem cell defect. In addition, evidence for residual stromal damage was evidenced by persistence of a maturational defect following "correction" of the aplasia in irradiated busulfan-treated mice. For over fifty years the bone marrow has been recognized as being particularly susceptible to radiation injury and a cause of aplastic anemia in mice. The dog has been used as a model for radiation-induced aplastic anemia figuring prominently in models used for bone marrow transplantation. Radiation produces both transient and prolonged bone marrow suppression, depending on the dose, dose rate, and exposure conditions. Local irradiation of rats results in a transient hypoplasia and recovery following 2000 rds, whereas twice the dose results in a transient hypoplasia, with a latent period followed by a prolonged hypoplasia. Aplastic anemia resulting from either busulfan or irradiation is characterized by a marked reduction in CFU-S, BFUE, CFU-E and CFU-GM in mice. Despite the unquestioned potential o f benzene to produce bone marrow damage in man, animal models of benzene-induced aplastic anemia remain difficult to produce. Experimental evidence has been contradictory, in some instances; however, examination of these findings in conjunction with those r previously described for the effects of benzene metabolites on macrophage function indicate that benzene exposure results in prolonged stem cell abnormalities, both abnormal differentiation and damage to the stromal cell popul ations. #3 HD Myelofibrosi s Following injury the bone marrow may undergo necrosis followed by replacement by fibrosis. This reaction may become progressive, resulting in virtually total displacement o f hematopoietic precursor cells with fibrous tissue. Myelofibrosis in man is now classified as a myeloproliferative disease according to the International C1 assification of Diseases. Based on studies of saponin-induced myelofibrosis in rabbits, it has been hypothesized that the pathogenesis of the disease predominantly involves interference with the blood supply to the bone marrow. Hematologic findings vary considerably, depending on the species and the severity of the lesion; however, the spleen is usually enlarged, with myeloid metaplasia a predominant feature. #2 HD Clonal Hemopathies Recognition of the existence and nature of the clonal hemopathies resulted from the integration of two independent 1 ines of experience in clinical and experimental hematology. Firstly, it has been recognized that leukemia in man is frequently preceded by progressive hematologic abnormalities (hematopoietic dysplasias), which represent a spectrum or continuum of abnormalities rather than separate or unrelated entities. Secondly, considerable evidence suggests these dysplasias, collectively termed P myelodyplastic syndrome (MDS), and leukemias are predominantly clonal hemopathies, sharing a common stem cell target. #3 HD Myelodysplastic syndrome (MDS) It has been recognized for nearly thirty years that cases of leukemia, primarily acute myelogenous leukemia, are often preceded by a "preleukemic" o r prodromal state of chronic bone marrow insufficiency. This phenomenon occurs with alarming frequency in leukemias arising secondarily to previous cancer therapy. A number o f studies have also shown that MDS often precedes lymphoproliferative disorders. MDS can be manifested as either bone marrow insufficiency or a pro1 iferative disorder. Insufficiency i s associated with varying degrees of anemia, usually accompanied by macrocytosis, and leukopenia or thrombocytopenia. Histologically, the bone marrow is usually hypocellular or an excessive proliferation of one or more cell lineages can be observed. The rate o f conversion of MDS t o frank leukemia in man appears to be particularly high i n cases arising secondary to chemotherapy or benzene exposure. Studies utilizing cytogenetic or biochemical markers to identify the clonal origins o f abnormal cell populations have revealed that clonal abnormal i ties may be present for years prior to the clinical onset of leukemia. MDS, itself carries a grave prognosis with mortalities reported of 30 - 84%. Experimentally induced MDS has been studied in dogs, rats and mice. Dogs receiving a leukemogenic dose of radiation developed acute nonlymphocytic leukemia preceded by changes identical to MDS in man. During the recovery c phase animals exhibited macrocytic anemia, thrombocytopenia and neutropenia. Bone marrow exhibited hypercellul arity and severe hematopoietic dysplastic changes. Persistent dysplastic myeloproliferative lesions have been reported in rats following treatment with dimethylbenz(a)anthracene, and in mice following exposure to benzene. In both species signs of hematopoietic dysplasia were most obvious in the spleen, consistent with its role as an extramedullary hematopoietic organ in rodents. Hyperplastic and dysplastic changes in the erythroid series were prominent in rats, whereas, myeloid dysplasia predominated in mice. A macrocytic anemia was observed in most mice, and hyperplastic changes in the spleen were prominent despite the presence of a persisting anemia. Abnormalities were noted in the number and morphology o f granulocytic, megakaryocytic and erythroid precursor cells, including nuclear deformities and atypical mitotic figures. #3 HD Leukemia \.c- Leukemias are among the most widely recognized and feared malignancies FII -in man. Derived from bone marrow, they are usually disseminated via the blood at some time during their development. In general, leukemias can arise as abnormal cell populations from any cell lineage, although there i s some predisposition to one or more cell types, depending on the method of induction and the species or strain studied. In the United States and Europe, leukemias - in man associated with chemical or drug exposure are predominantly myeloid, although secondary lymphoid leukemias and lymphomas are by no means rare. -. With the exception of certain strains, eg. CBA/Ca; mice exhibit a r predisposition toward lymphoid neoplasms. Spontaneous leukemias or viral- induced have been described in virtually every mammalian species examined. A comparative discussion of spontaneous leukemias across species i s beyond the scope of this chapter. Because of the singular prominence of the rodent in chemical leukemogenesis studies, the focus here will be on the rat and mouse. The abnormal cell population tends to be arrested at a single stage of differentiation in most leukemias and lymphomas. A1 though there are exceptions, leukemias consisting predominantly of immature or blast cells tend to proliferate rapidly and be aggressive, whereas those i n which a more differentiated phenotype prevails, tend to take a more protracted course. A common feature of hematologic malignancies in both experimental animals and man is their clonal nature, i.e. they are derived from a single cell. Lymphoid and myeloid stem cells are consistently involved as the targeted cell compartment in leukemias o f lymphoid and myeloid origin. As a result, the biologic significance concerning differences among acute lymphoid and myeloid leukemias amongst species, may prove to be less important than were historically assumed. On the other hand, chronic myelogenous leukemia in man, originates in an earlier cell, perhaps the PSC. It is apparent, therefore, that elucidation of the mechanism(s) of leukemogenesis i s inextricably linked to an understanding of stem cell regulation. Leukemogenesis is a multi-factorial process which cannot be modeled or simulated as a function o f a uni- or a bimolecular event. Therefore, it is not surprising that no initiation-promotion paradigms have been established for chemical leukemogenesis in experimental animals. #4 HD Y Diagnosis Leukemia and/or lymphoma has been defined as "[increased] number of specific hematopoietic cells in the absence of a demonstrable, appropriate stimulus. Cellular excess may be localized to a single lymph node, ... or may be quite widespread and involve bone marrow, blood and many lymph nodes as well as other organs.. . ' I 4 , This definition belies the difficulty which often obscures the diagnosis of leukemia, especially in rodents which constitute the most frequently used models in experimental carcinogenesis. The importance o f distinguishing between leukemias and lymphomas and other "non-malignant" dysplasias of the hematopoietic system has been exaggerated in government regulatory policy, where an almost mystical significance appears to be attached to the diagnosis o f leukemia. It should be noted that the biological significance of a differential diagnosis o f myeloid leukemia versus myelodysplasia to the host is virtually nil. The distinction between lymphoma, a malignancy involving primarily the thymus, lymph nodes o r splenic white pulp, and leukemia, a malignancy involving primarily the bone marrow, spleen or blood is often difficult or unwarranted. Mouse lymphoid neoplasms form space occupying 1esions, yet spread in a leukemoid manner, rendering the distinction difficult. Witnrobe, 1974 - Granul ocytic neoplasms usual 1y present as bl ood borne neoplasms in most species, whereas they exhibit a significant tendency to spread as solid tumors in rodents. While these distinctions may be of little biological significance, they can be important considerations in the interpretation of c tumor incidence data derived from animal studies in which different classification schemes are used. #5 HD Rat Spontaneous leukemias are rare in most strains of laboratory rat. This species is also relatively resistant to chemically-induced leukemogenesis. Both lymphoid leukemia and chloroleukemia have been reported in Wistar rats following the administration of methylcholanthrene. A1 though spontaneous granulocytic leukemias have been reported in Sprague Dawley rats, they are rare. Large granular cell leukemia (LGL) arises i n 10 - 50% of Fischer 344 rats over 18 months of age limits their usefulness in lifetime bioassays. First recognized and described as a specific entity in Wistar/Furth and Fischer rats, this neoplasm constitutes a major cause of death in F344 rats 2 years of age or older. Compounds, such as allyl isovalerate and ethylene oxide, have been demonstrated to increase the incidence of LGL leukemia in F344 rats; however, controversy e x i s t s as to whether this increase represents a leukemogenic response or an a1 tered onset of a spontaneously occurring tumor. Morpho1ogically, LGL cells resemble 1arge granular lymphocytes and have been identified as natural killer cell of the rat. The neoplasm arises in the spleen and spreads to the bone marrow late in the course of the disease. LGL i s accompanied by splenomegaly and an autoimmune hemolytic - anemia. Erythrophagocytosis is a common feature in LGL infiltrates in the spleen. #5 HD P Mouse Many classification schemes o f murine lymphoid neoplasms have been described. One of the most widely adopted classifications is that proposed by Dunn' which separates lymphoid neoplasms into lymphocytic, reticulum cell types A and 8, and plasma cell lymphomas, based solely on morphology. Pattengale and Taylor6 introduced an immuno-morphologic classification of murine lymphomas. Both o f these schemes provide detailed criteria for the diagnosis of histocytic and 8-cell lymphomas, which constitute the vast majority of spontaneous lymphomas encountered in most mouse strains. In contrast, thymic (T cell) lymphoma/leukemia occurs rarely in most murine strains. The AKR and C58 mouse strains are principal exceptions in which thymic lymphoma appears spontaneously within the first year of life and is closely associated with endogenous ecotropic retrovirus. Thymic lymphoma i s the principal neoplasm associated with radiation or xenobiotic exposure in a variety of strains, including C57BL/6,, RF, and C3H mice. The respective roles of chemical exposure and retroviruses in these murine models remains the subject of debate. Regardless of the classification scheme employed, care must be taken to use comparable diagnostic criteria when comparing the results o f different ' Dunn, 1954 `Pattengale and Taylor, 1981; 1983 - studies. The B6C3F1 hybrid (C57BL/6 x C3H) mouse has become the standard murine model for chemical toxicity and carcinogenicity studies, because of the low incidence of spontaneous lymphomas encountered in this hybrid, relative to other mouse strains. Nevertheless, over a two year lifespan, the incidence o f P spontaneous lymphoid neoplasms is 8.3% in males and 16.8% in females. These tumors arise late in life, most frequently in lymph nodes, spleen or liver, and are presumably of B-cell origin. The incidence of spontaneous thymic lymphoma in these mice is negligible. The incidence of lymphoma in B6C3F1 mice chronically exposed to 1,3-butadiene is high, approaching 60% in males by the end of one year. In contrast to spontaneous lymphomas, these tumors are mediastinal in origin and uniformly express T-cell surface markers. A1 though butadiene would be classified as a murine leukemogen, it is appropriate t o discuss thymic lymphomas in the context of a negligible historical incidence. To date, few chronic studies have employed sufficient diagnostic criteria to make such distinctions. Differentiating hyperplastic versus neoplastic lymphoid lesions is difficult, particularly in mice, as tissue availability is 1 imited. Conservative criteria should be used, since normal lymphoid populations possess some characteristics suggestive of malignant cells in sol id tissues; normal lymphoid populations retain the capacity to divide, have the ability t o invade lymphoid and non-lymphoid tissues, and can kill targeted cells. As a result, reactive hyperplastic responses can be extremely difficult to distinguish from truly malignant neoplasms by histopathologic analysis alone. When in doubt, successful transplantation of tumor cells into syngeneic or nude hosts remains the sine qua non for demonstrating malignancy. The diagnosis of murine myeloid leukemia is also difficult, as myeloprol iferative disease i s characterized by features often commonly encountered in both neoplastic and acute inflammatory processes. Under conditions of acute inflammation, or hematopoietic stress, the spleen is the c first site of extramedullary hematopoiesis. It is also frequently the primary site for the development of myelogenous leukemia. To confuse matters further, both processes can be accompanied by extensive myeloid hyperplasia in the red pulp of the spleen. The early infiltration into surrounding tissues by leukemic cells is similar to that encountered in extramedullary granulopoiesis in response to acute inflammation. Various authors have differed widely in their criteria for diagnosis of leukemia in mice. Dunn7 suggested using the lack of a local inflammatory reaction and the presence o f collections of relatively immature granulocytic or blast cells as diagnostic criteria, particularly in areas where extramedullary hematopoiesis is seldom found. Unfortunately, neoplastic proliferation in the mouse is often characterized by a variable range of maturation in the myeloid series, so Barnes and Sisman' concluded that a predominance of blast cells and ring forms are very reliable criteria for distinguishing neoplastic from compensatory hyperpl asia. Although total replacement of lymphoid follicles is a distinguishing feature in cases o f myeloid leukemia, some follicular remnants are almost always spared. Because murine myeloid tumors exhibit a tendency to spread I Dunn, 1954 Barnes and Sisman, 1939 - either by direct extension or emboli, and acute inflammation is accompanied by a marked neutrophilia in mice, leukocytosis cannot be considered a reliable diagnostic sign. In cases where the disease is largely limited to the spleen, the predominance of immature cells in conjunction with intravascular extension r or spread may be an important morphologic differentiating factor. In the final analysis, the distinction between a malignant and non-malignant myeloproliferative lesion is definitely not black and white. #1 HD Methodology for the Evaluation o f Bone Marrow Toxicity Increasingly sophisticated automated methods f o r the identification of individual cells, long term bone marrow cell culture, and technologies for cell and molecular cloning have led t o an explosion the number of methods available for the study of toxicity to the hematopoietic system. The purpose of this section is to provide a general discussion o f the advantages and potential limitations o f various approaches used to evaluating toxicity to the blood and bone marrow. Detailed discussions o f individual methods and appl ications are readily available el sewhere in the 1 iterature. #2 HD #3 HD Eva1uation o f Toxicity C1inical Laboratory Hematology Analysis of the blood remains the most widely employed method for evaluating hematotoxicity o f chemicals and physical agents. Most procedures commonly employed in the human clinical laboratory have been adapted for use in experimental animals. The complete blood count (CBC) typically includes: the leukocyte count (WBC), erythrocyte or red cell count (RBC), hemoglobin (HGB) , hematocrit or packed cell volume (HCT or PCV) , platelet count (PLT), and erythrocyte indices, ie. mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), morphologic examination of the peripheral blood smear, and differential P leukocyte and reticulocyte counts. With more advanced techno1ogy, parameters such as the red cell distribution width (RDW), are now available. Procedures used for the diagnosis of human clotting disorders have also been adapted for use in experimental animals. Descriptions of both manual and automated methods, and standardization of these procedures, are available in any hematology reference. Most commercially available instrumentation for automated analysis of blood can be adapted for experiments which often have small samples. The limitations o f small sample size cannot be overemphasized when using rodents, especially mi ce! Sampling site and collection method influence hematology values; therefore, site i s another important consideration in the design, interpretation and comparison of hematologic studies in rodents. Sampling sites can be a major source of variation between experiments. Sources commonly used include: tail, orbital plexus, abdominal aorta, abdominal vena cava, and heart. Tail blood WBD are approximately twice that of abdominal aorta and significantly higher than those from the orbital plexus. Although the orbital plexus represents a frequent site for serial sampling of blood from rodents, it should be recognized that such samples are primarily capillary in origin and differ from other sites with respect to WBC, RBC, HGB, L HCT and differential counts. Furthermore, samples taken from the orbital plexus vary depending on the operator's technique. In addition site having an effect on hematologic parameters, it also r important in determining what volume can be collected. The order of collection can account for differences; hematology samples should be drawn first. In general, hamatological parameters measured from right ventricular blood tend to be the least variable and most consistent. #3 HD Bone Marrow Cellul arit y Bone marrow cellularity, being relatively independent of the transient fluctuations in blood leukocytes and platelets, i s a useful adjunct to the complete blood count. Quantitative methodology is considerably more sensitive than histopathologic analysis. It i s possible to detect changes of 25% o f a certain cell type using quantitative methodology, whereas a change of 80% or more is required for reliable detection by histopathology. A variety o f procedures quantify bone marrow cellul arit y including : hemocytometers, automated cell counters, and the use of 59Fe uptake. Cellularity i s usually expressed in terms of the total number o f nucleated cells per milligram, in rats or larger animals; the total number o f cells per tibia or femur can be determined in mice. #3 HD F1 ow Cytometry Techniques such as flow cytometry, in conjunction with modern cell and molecular biology, have revolutionized experimental and clinical hematology. Flow cytometry allows high speed multiparameter analysis and purification of individual cells in suspension. Fluorescence detection methods have been successfully applied to DNA and RNA/DNA for cell cycle analysis, cytotoxicity, phagocytosis, and cell identification (using surface marker analysis). Cell c sorting based on surface marker analysis has recently been coupled with mechanical cell purification technology for the successful purification of murine PSC. #3 HD Ferrokineti cs Ferrokinetic analysis? measuring 59Fe uti1 ization, has occasionally been used to monitor chemical toxicity to the bone marrow in rodents. It i s a crude indicator of bone marrow function. Nevertheless, ferrokinetic data in rodents must be interpreted with caution for a variety o f reasons: Firstly, incorporation of iron into erythroid precursors is not uniform. Uptake is greatest in immature erythroid cells and diminishes with progressive differentiation. Secondly, the use of ferrokinetic data to estimate the differential effects of chemical toxicity on various erythroid precursor compartments assumes a constant hematopoietic tissue mass. This is a problem which cannot be control1ed for in rodents because of extramedullary hematopoiesis. Thirdly, under non-steady state conditions the number of divisions necessary to produce reticulocytes may vary. Finally, the premature loss of erythrocyte precursors, i e . "ineffective erythropoiesis", may vary with toxicity or proliferative response t o blood loss. #2 HD #3 HD Analysis o f stem cell populations. CFU-S The traditional in vivo mouse spleen-colony forming assay (CFU-S) w remains the standard method to quantitatively assess PSC in mice. The assay assumes that multipotential stem cells will give rise to colonies of hematopoietic cells in the spleen of lethally irradiated mice, following inoculation with a limiting number of bone marrow cells. Morphologic evaluation of these colonies reveals erythrocytic, granulocytic, megakaryocytic, or mixed cell type colonies. Mixed cell colonies are indicative of a multipotential precursor cell. The CFU-S assay is essential for the initial evaluation of stem cell alterations. Nevertheless, the assay possesses certain 1 imitations that must be considered when applying it to the evaluation of chemical toxicity. Firstly, timing of the assay procedure is important. Colonies are apparent in the spleen seven days after reconstitution; however, only colonies forming after 10 - 12 days correlate well with PSC activity. Because survival of lethally irradiated mice beyond 8 days is difficult in the absence of effective barrier facilities, some investigators have chosen to evaluate the effects of chemical pretreatment on CFU-S 8 days after inoculation. Unfortunately, colonies appearing at 8 days post-inoculation represent a disproportionately high percentage of committed erythroid progenitor cells, which more than likely may disappear with time; these do not measure PSC. Secondly, it is now recognized that the PSC compartment itself is heterogeneous, ranging from cells with a high potential for self-replication and less propensity to differentiate (CFU-S-l), to those with relatively limited ability to replicate and a high tendency to differentiate (CFU-S-11). These represent two major subsets of the PSC compartment that are presumably affected by exposure to myelotoxic agents and that are not readily distinguished using the in vivo CFU-S assay. Thirdly, the CFU-S assay has 9 limited sensitivity, with approximately 5% of injected stem cells colonizing the spleen. Fourthly, the maximum number of colonies that can be reliably distinguished in the mouse spleen before confluence of colonies obscures results is on the order of 12-15. Lack of distinction, coupled with the high mortality in lethally irradiated mice, results in the requirement for large numbers of recipient animals and/or the use of pooled donor cells, which limits experimental design. Finally, the seeding efficiency o f active PSC is less than half that o f resting PSC, rendering interpretation of CFU-S data difficult in the face of altered stem cell activation resulting from chemical exposure. Thus, the representative subset of PSC measured by CFU-S during a compensatory hematopoietic response to bone marrow injury is probably a1tered over and above a treatment-related effect on the absolute number of PSC. These characteristics of the assay can serve to limit its usefulness or, alternatively restrict the interpretation that can be placed on CFU-S data when used to evaluate mechanisms of stem cell injury. #3 HD CFU-Mix The in vitro multi-CFC assay is an alternative method which offers some advantages including: increased sensitivity, elimination of interpretive problems related to changes in stem cell splenic colonization efficiency, and elimination of the requirement for using large numbers of lethally irradiated mice. Unfortunately, the potential for self renewal in multi-CFC populations - appears t o be extremely low. I t i s l i k e l y t h a t t h e major c e l l type enumerated i n t h e CFC mix assay corresponds t o CFU-S-11, b u t i t may be f e a s i b l e t o d i r e c t l y d s c r i m i n a t e between CFU-S-I and I 1 i n the i n v i t r o a s s a y . An advantage of i n v i t r o bone marrow c u l t u r e systems i s the p o t e n t i a l f o r d i r e c t r comparison of animal c e l l s and/or w i t h human stem cell p o p u l a t i o n s - a c a p a b i l i t y obviously not afforded w i t h the use o f the mouse CFU-S assay. #3 HD Progenitor cell assays C l o n i g e n i c assays have been developed t o measure v i r t u a l l y every known progenitor c e l l population, and provide a means t o measure the effects of a g e n t s on p r o g e n i t o r cell numbers following exposure i n vivo. A1 t e r n a t i v e l y , t h e y can be used t o e v a l u a t e the e f f e c t s of i n v i t r o exposure t o myelotoxic a g e n t s on p r o g e n i t o r cell d i f f e r e n t i a t i o n and pro1 i f e r a t i o n . The use and measurement of defined growth f a c t o r s makes e v a l u a t i o n of the r o l e s of growth f a c t o r elaboration and t a r g e t cell response i n myelotoxicity a p o s s i b i l i t y . #3 HD Long Term Bone Marrow Cultures Techniques and conditions have a l s o been defined t h a t enable the long term culture of pl u r i p o t e n t i a1 and p r o g e n i t o r bone marrow c e l l s . Pioneered by Michael Dexter and c o l l e a g u e s i n the l a t e 1970's9, these methods involve the co-cul ture o f hematopoietic and adherent bone marrow stromal cells, c r e a t i n g c o n d i t i o n s a t l e a s t p a r t i a l l y analogous t o the in microenvironment. Studies using this technique have revealed t h a t t h e adherent layer is the 'Dexter d.,1982 - major site of stem cell origin, which subsequently releases progenitor and differentiating lineage-committed precursor cells . Although the re1ati onships between adherent stromal cells , stem, progenitor cells and their defined growth requirements are necessarily complex in these co-culture P systems, they offer considerable promise f o r the study o f the mechanisms of drug and chemical action on regulation of hematopoiesis. #1 HD SUGGESTED READING # 2 HD Gross and Microscopic Anatomy Lichtman,M.A. (1981). The ultrastructure of the hemopoietic environment o f the marrow. A review. EXP. Hematol. 9, 391-410. Weiss, L.*(1968). Structure o f the lymphatic apparatus. Cancer Chemother. Reports. 52, 21-28. Weiss, L. (1980). The haemopoietic microenvironment o f bone marrow: An ultrastructural study of the interactions of blood cells, stroma and blood vessels. In "Blood Cells and Vessel Walls: Functional Interactions" Ciba Foundation Symposium 71, pp. 3-20. Excerpta Medica, Amsterdam. #2 HD Hematopoeisis Stem and Progenitor Cells Abramson, S., Miller, R.G., and Phil1 ips, R.A. (1977). The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J.Exp.Med. 145, 1567-1579. Axelrad, A.A., McLeod, D.L., Shreeve, M.M., and Heath, D.S. (1974). Properties of cells that produce erythrocytic colonies in vitro. In "Hemopoiesis in Culture" (W. Robinson, ed.), p . 226. US Gvt. printing office, Washington, D. C. Barnes, D.W.H., Ford, C.E., Gray, S.M., and Loutit, J.F. (1959). Spontaneous and induced changes in cell populations in heavily irradiated mice. Geneva Conference Paper. 2, 1-10. Becker, A.J., McCulloch, E.A., and Till, J.E. (1963). Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature. 197, 452-454. Calvo, W . , Fliedner, T.M., Herbst, E., Hugl, E., and Bruch, C. (1976). Regeneration of blood forming organs after auto1ogous 1eukocyte transfusion in lethally irradiated dogs. 11. Distribution and cellularity of the marrow in irradiated and transfused animals. Blood. -47, 593-601. Downey, H. (1938). "Handbook o f Hematology". P.B. Hoeber Inc. New York. Fauser, A.A., and Messner, H.A. (1979). Identification of megakaryocytes, macrophages and neosinophils in colonies o f human bone marrow containing neutrophilic granulocytes and erythroblasts. Blood. 53, 1023-1030. Fialkow, P.J., Denman, A.M., Jacobson, R.J., and Lowenthal, M.N. (1978a). Chronic myelocytic leukemia: origin of some lymphocytes from leukemic stem cells. J. Clin. Inv. 62, 815-849. Hara, H., and Ogawa, M. (1978). Murine hemopoietic colonies in culture containing normoblasts, macrophages, and megakaryocytes. Am. J. Hematoloqy 4, 23-34. Jacobson, L.O., Marks, E., and Gsaton, E. (1954). Observations on the effect of spleen shielding and the injection of cell suspensions on survival following irradiation. Radiobioloqv Svrnoosium. 122-133. t. Johnson, G.R., and Metcalf, D. (1977). Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythroprotein. Proc. Nat. Acad. Sci. 74, 3879-3882. Johnson, G.R., and Nicola, N.A. (1984). Characterization o f two populations of CFU-S fractionated from mouse fetal 1 iver by fluorescence-activated cell sorting. J. Cell Phvsiol. 118,45-52. Lemischka, I.R., Rauler, D.H., and Mulligan, R.C. (1986): Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 45, 917-927. Magli, M.C., Iscove, N.N., and Odartchenko, N. (1982). The transient nature of early haemopoietic spleen colonies. Nature 295, 527-529. Maximow, A.A. (1924). Relation o f blood cells to connective tissue and endothelium. Phvsiol. Rev. 4, 533-563. Messner, H.A., and Fauser, A.A. (1980). Culture studies of human plruipotent hemopoietic progenitors. Blut. 4 l , 327-333. Messner, H.A., Jamal, N., and Izaguirre, C. (1982). megakaryocyte colonies from human bone marrow. The J. gCerlolwthPhovsfiollar.gSeu w l . -1, 45-51. Metcalf, D., Johnson, G.R., and Mandel, T.E. (1979). Colony formation in agar by multipotential hemopoietic cells. J. Cell Phvsiolosy,98(2), 401-420. Metcalf, D . , Johnson, G.R., and Nicola, N.A. (1982). Separation and kcommitment o hemopoietic stem and progenitor cells, In "Haemopoietic Stem Cells" illman, SV- AA., Cronkite, E.P., Muller-Berat, C.N. eds.), pp. 29-38. Munksgaard, Copenhagen. Molineux, G., Schofield, R., and Testa, N . Q (1986). Development of spleen CFU-S colonies from Day 8 to Day 11: relationship to self-renewal capacity. EXD. Hematol. l4, 710. Nakahata, T., and Ogawa, M. (1982). Identification in culture of a class of hemopoietic colony-forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies. Proc. Nat. Acad. Sci. -79, 3843-3847. Patt, H.M., and Qaustler, H. (1963). Radiation effects on cell renewal and nrelated systems. Physiol. Rev. 43, 357-396. - Simminovitch, L., Till, J.E., and McCulloch, E.A. (1963). Decline in colonyforming abil ity of marrow cells subjected to serial transplantation into irradiated mice. 3. Cell and Como. Phvsiol. 64, 23-32. Snodgrass, R., and Keller, G. (1987). Clonal fluctuation within the haematopoietic system of mice reconstituted with retrovirus-infected stem cells. The EMBO J,6(13), 3955-3960. P Spangrude, G.J., Heimfeld, S., and Weissman, I.L. (1988). Purification and characterization o f mouse hematopoietic stem ce7ls. Science 241, 58-62. Till,J.E., and McCulloch,E.A. (1961). A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Res. l4, 213222 L van Bekkum,D.W. (1977). The appearance o f the mu1 tipotential hemopoietic stem cell, In "Experimental Hematology Today" (S.J. Baum and G.D. Ledney, eds.), p. 3 . Springer Verlag, New York. Visser,J.W.M., Bauman,J.G.J., Mulder,A.H., Eliason,J.F. and deLeeuw,A.M. (1984). Isolation of murine pluripotent hemopoietic stem cells. J.ExD.Med. 59, 1576-1590. Weed,R.I. (1970). The importance of erythrocyte deformability. Am.J.Med. 49, 147- 150. Wilson,F.D. (1985). Clonogenic Stem and Progenitor Cell Assays for the evaluation o f chemically induced myelotoxicity, In "Toxicology of the Blood and Bone Marrow" (R.D. Irons, ed.), pp. 65-100. Raven Press, New York. Wu,A.M., Till,J.E., Siminovitch,L., and McCulloch,E.A. (1967). A cytological study o f the capacitiy for differentiation of normal hemopoietic colonyforming cells. 3. Cell. Phvsiol. 69, 177-184. #2 HD Regulation o f Hematopoiesis. Bagby,G.C.Jr., McCall,E., and Layman,D.L. (1983). Regulation of colony stimulating activity production: Interactions o f fibrobl asts, mononuclear phagocytes and lactoferrin. J.Clin.Invest. 7 l , 340-344. Bonsdorff,E., and Jalavisto,E. (1948). A humoral mechanism in anoxic erythrocytosis. Acta Physiol.Scand. l6, 150-170. Broxmeyer,H.E. (1986). Biomolecule-cell interactions and the regulation of myelopoiesis. Int.J.Cell C1 oninq Q, 378-405. Broxmeyer,H. E., Wi 1 1 i ams,D.E., Hangoc,G., Cooper,S., Gi11 i s, S., Shadduck,R.K., and Bicknell ,D.C. (1987). Synergistic myelopoietic actions in vivo after administration to mice o f combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3 , and recombinant murine granulocyte/macrophage colony stimulating factor. Proc.Nat.Acad.Sci. 84, 3871-3874. Burgess,A.W., Metcal f,D., Russel ,S.H.M., and Nicol a,N.A. (1980). Granulocyte/macrophage- , megakaryocyte-, eosinophi1 - and erythroid colony-stimulating factors produced by mouse spleens. 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Clin.Invest. 77, 1857-1863. - Table 1 Hematopoietic Growth Factors Growth Factor Synonyms Source Target Cells - Known Activity ---------r-----,------------------------------------------------- IL-3 Mu1ti -CSF T-lymphocytes Supports growth o f early progenitor cells: CFUs, CFC-Mix, pre- lymphocytes, macrophage progenitors and mast cells. Acts synergistically with Epo or G-CSF to stimulate BFU-E and CFU-G. M-CSF CSF- Endothel i a1 cells, macrophages Mouse L cells Supports growth o f macrophage progenitors M-CSF CSF-2 Endothe1 ia1 Macrophages lung cells Supports macrophage and granulocyte progenitors. Acts synergistically with other CfaFcUt-Moergs.to support BFU-E, CFU-G and G-CSF T lymphocytes macrophages Stimulates CFU-G. Acts synergistically with IL-3 and GM-CSF to support CFU- and CFU-M. Erythropoieti n Kidney Stimulates CFU-E and proerythrobl asts. Figure 1. - Schematic diagram of hematopoiesis, showing the relationship between pro1 iferation and sel f-renewal in the stem cell and precursor cell compartments. - Figure 2. c Figure 3. Kinetics of granulocyte formation illustrating the relatively small proportion of granulocyte pool in circulating and marginated compartments (Cronkite, 1969). (a) DNA cell cycle based on hypothetical 24 hr. transit time. (b) Phases of the cell cycle as defined by relative DNA content o f cells. Figure 4. DNA cell cyle histogram obtained by flow cytofluorernetric analysis o f asynchronously dividing rabbit bone marrow cells. (Irons and Stillman, 1985). Figure 5. .(a) Photomicrograph of normal rabbit bone marrow showing normal cell ul ari ty (b) Hypopl ast i c bone marrow foll owing 10 days repeated benzene precursor cell s. exposure. Note vertical (Methacryl ate: Toluidine 6a1bsuee)nc.e of viable