Document 0q0jOeg4dKJ0zkdv002vooZ7x

Assessment of Myelotoxicity Caused by Environmental Chemicals by G. A. Boorman,*1 M. I. Luster/ J. H. Dean* and 1 M. L. Campbell* I < Potential antineoplastic agents must be screened for the delayed toxicity that occurs in many j eases of drug-induced bone marrow aplasia. In vitro clonal assays for hematopoietic progenitor I cells have been developed to assess the degree of myelotoxicity. This adverse side effect is often j the limiting factor in the development of new cancer chemotherapeutics. In addition, many environmental chemicals are cytotoxic to rapidly proliferating cells, but a systematic assessment j of their myelotoxicity has not been performed. We have used clonal marrow assays to investigate I a panel or chemicals including 2,3,7,8-tetrachIorodibenzo-p-dioxin, polybrominated biphenyls, J diethylstilbestrol, benzofalpyrene and indomethacin. All were immunotoxic, some to pleuripotent I hemopoetic stem cells and others to granulocyte-macrophage progenitors, and at concentrations below those causing other toxic manifestations. This shows that these bone marrow clonal assays, and hopefully future ones for erythroid, B-and T-lymphocytes, and megakaryocytes, will provide the specificity and sensitivity necessary to delineate the myelotoxicity of a broad spectrum of environmental chemicals. Introduction , As one of the most rapidly renewing cell populahemopoietic cells have proven very sensitive to cytotoxic agents (1-5). Bone marrow failure is a puficant complication of cancer chemotherapy and also been implicated as a result of exposure to drugs (6-9) and environmental agents UO-12). During the past decade, a variety of semim vitro culture techniques have been devel oped for most of the hemopoietic cell lines. *&ounation of colony formation of the hemopoietic ^ following exposure to various agents has prov'* to be sensitive indicator of toxicity as web as a mechanisticstudy of the toxicity ofvarious jJll&Wal Toxicology Program and Laboratory of EnvironChemistry, National Institute of Environmental Health -> National Institutes of Health, P.O. Box 12233, Relark. North GcnLina 27709 to whom reprint requests should be addressed. 1982 Murine pleuripotent hemopoietic stem cells can be detected by injecting bone marrow cells into irradiated recipients and counting the number of colonies that form in 8 days (IS). More differentiat ed committed progenitor cells can be induced to form colonies in semisolid media by adding appro priate stimulatingfactovs. Currently, clonal assay procedures exist (Fig. 1) for B- and T-lymphocytes, macrophage-granulocyte progenitors, megakaryo cytes, eosinophils and erythroid precursors (U,15). Colonies arise from proliferation of individual cells and the number of colony forming cells are altered under abnormal physiological conditions following treatment with certain drugs, and in certain dis ease states such as chronic myelogenous leukemia and polycythemia rubra vera (15). Hemopoietic stem cells appear to have a limited capacity for division. This can be demonstrated by serial`passage of bone marrow cells into irradiated recipients (16). Serial transfer of the donor cells will permit the marrow tissue to survive beyond the normal life expectancy and shows that bone mar- 129 Lymphocytes 8FU-E CFU-E Erythrocytes Monocutes neutrophils Figure 1. Model of hematopoiesis as determined by in vitro and in vivo clonal assays. CFU-S, colony-forming unitspleen; CFU-TL and CFU-BL, colony-forming unit-T and B-lymphocyte, respectively; BFU-E, burst-forming uniterythroid; CFU-E, colony formingumt-erythroid; CFIJ-GM, colony-forming unit-granulccyte-macrophage; CFU-M, colony forming unit-megakaryocyte; CFU-Eo, colony-forming uniteosinophil. Figure 2. Scheme of testing for myelotoxicity. Following ex posure to a chemical a single cell suspension is made of femoral marrow. Following enumeration of nucleated mrow cells they are injected into irradiated recipients to me*, sure colony-forming units-spleen (CFU-S) and plated in in vitro cultures to which has been added colony-stimulating factor (CSF), erythropoietin (EPO) or pokeweed stimulated lymphocyte culture medium (PSLCM) which will allow the proliferation ofgranulocyte-macrophage progenitors (CPU-GM), erythroid progenitors (BFU-E and CFU-E) and megakaryo cytes (CFU-M), respectively. row would not be a limiting factor in aging of the mouse. Fetal or embryonic hemopoietic tissue has a greater capacity for serial transfer than from adult mice. However, serial transfer capacity changes very little with aging, suggesting the stem cell compartment is minimally used during adult life (28). Several studies have shown that exposure to a toxic agent such as busulfan can induce a perma nent stem cell defect that persists throughout the life of the animal without any evidence ofrecovery (19,20). Animals with permanent stem cell defect may have normal bone marrow cellularity and pe ripheral blood counts, but the residual injury can be demonstrated by increased sensitivity to irradia tion (19), decreased erythropoietic response follow ing anemia stress (21), decreased proliferative capacity of bone marrow or after serial transplanta tion (22) and decreased antibody response (20). These animals wi 11 eventually die of chronic aplastic marrow failure (28). The concept of residual marrow injurymay prove to be very important in toxicology testing. Current ly toxicological assessment devotes little effort to understanding myelotoxicity and the usual screen ing programs would not detect residual stem cell injury since these animals may appear clinically normal. Chronic aplastic marrow failure, frequent ly a fatal syndrome, has a history of drug or chemi cal exposure in approximately 50% of the cases (28). Although marrow failure is rare, the studies cited 130 above suggest a much larger population may exist with undetected residual stem cell injury following chemical exposure. This population would show decreased ability to respond to situations requiring increased hemopoietic cell production and may be more susceptible to other cytotoxic drugs. While the model of induced stem cell defect and marrow failure was developed in mice, it would appear relevant to man, since it has been shown that patients exposed to chemotherapy show a prolonged decrease in bone marrow colony-forming cells in spite of normal peripheral blood counts, and it has been suggested that the marrow is more fragile in these patients (24). Based on the mouse model, these patients would be expected to have less bone marrow reserve when extra hematopoietic cells are needed (Fig. 2). Assessment of Bone Marrow Capacity by Use of Clonal Assays Pleuripotent Stem Cells (CFU-S) 'flu and McCulloch (18) showed that when mouse marrow cells were injected into heavily irradiated Environmental Health Perspective* dents, discrete nodules of hemopoietic cells form in the spleen of the recipients in 8 or 9 Ays- The Modules, called spleen colonies, contained or mixed populations of various hemopoietic AS lints (15) ana were shown by chromosomal Barkers (25) to arise from individual cells which 1 hive been called colony-formingunit-spleen (CFU-S), or pleuripotent stem cells. When isolated, made into a suspension and reinjected into an irradiated recipient, a stem cell colony containing cells of one hemopoietic cell line would give rise to new colonies of all cell lines, demonstrating both the self-renewal capacity and pleuripotentianty of the CFU-S. The relationship of the CFU-S to lymphocytes, an area of controversy, sow appears to be resolved with the CFU-S and lymphoid progenitors being derived from a com mon, note primitive, stem cell (15,26). Most CFU-S have a long generation time or remain longer in the G# cell cycle phase. When marrow depletion occurs, / CFU-S respond by more rapid proliferation and differentiation. As mentioned^ earner, CFU-S have i finite self-replication capacity which under normal circumstances exceeds the life span of the mouse. Chemotherapy or irradiation can damage the repli cative capacity of CFU-S with bone marrow failure * a ]abs sequela. Granulocyte-Macrophage Progenitors (CFU-GM) Shortly after the development of the CFU-S assay, Bradley and Metcalf (27) as well as Pluznik nd Sachs (28) demonstrated that bone marrow aBb would, with appropriate stimuli, form discrete colonies in semisolia media in vitro. These colonies rose from individual cells, and the cells giving rise to .these colonies were designated colony-forming nts in culture or CFU-C. The proliferation of toese cells requires the presence of a colonystimulating activity (CSF), and in 7 days colonies containing both granulocytes and monocytetnacrophages are formed. A single cell can give rise t both granulocytes and macrophages (29), and "A cell has subsequently been renamed CFU-GM j* deny-forming unit, granulocyte-macrophage. TTM CFU-GM is a separate cell population from the -FU-S, has a relatively high proliferation rate and capacity for self-renewal (15). Conditions that r***n<i granulopoiesis result in increased serum tissue CSF Tevels as well as increased marrow VfG-GM numbers while cytotoxic agents cause CFU-GM numbers (SO). The CFU-GM ii available for most mammalian species inaonflr man and has led to the identification of *^Wary 1982 factors having both positive and negative effects on granulopoiesis. Erythroid Progenitors (CFU-E, BFU-E) More recently, it has been reported that the addition of erythropoietin to bone marrow cultures will stimulate the growth of colonies that contain hemoglobulin (31). Further study has shown that these erythroid colonies contain at least two classes of stem cells, a more primitive erythroid burst forming unit (BFU-E) requiring long culture peri ods (10-14 days) with high erythropoietin levels and a more differentiated colony-formingunit-erythroid (CFU-E) requiring lower erythropoietin level and only 2 days of culture (23,33). Marrow CFU-E numbers follow a predictive pattern, increased fol lowing bleeding (34) and decreased during plethora induced by red cell transfusions (35). Erythroid progenitors as measured by clonal assays are sensi tive to cytostatic agents (30,36,87) and known mar row toxicicants such as benzene (38,39). Use of these assays has revealed that some'patients with aplastic anemia possess a population of lympho cytes capable of suppressing in vitro erythroidcolony formation by normal human bone marrow (40), whereas in congenital hypoplastic (DiamondBlackfan) anemia, lymphocytes are normal, and the defect appears to reside in the erythroid progeni tors (41). Thus these assays allow not only study of erythroid progenitor cell numbers but can also con tribute to our understanding of hormonal factors and cell to cell interaction as may occur in disease states. Another technique used to study erythropoiesis is S9Fe uptake in spleen and marrow which provides aspecificmarker for hemoglobin synthesis (15,36). In this assay, mice are killed 18 hr after injection of the isotope and tissues collected for counting. Further studies are needed, however, to determine the correlation between 59Fe uptake and the clonal assays for erythroid precursors. Other Clonal Bone Marrow Assays (CFU-BL, CFU-TL, CFU-M) Clonal marrow assays have been developed for B-lymphocytes (CFU-BL),T-lymphocytes (CFU-TL), and megakaryocytes (CFU-M) by adding appropri ate stimuli to semi-solid marrow cultures (15,38-40). These new model systems should allow further understanding of granulopoiesis and may represent sensitive indicators of toxicity but need to be fur ther examined. 131 Effects c Environmental Agents on Myelotoxicity Benzene is one of the more widely known myelotoxic agents where short term exposure will induce profound pancytopenia and anemia Following benzene exposure, there is decreased uptake by hematopoieticorgansU-Z), but whether this compound acts at the more primitive BFU-E or the more mature CFU-E has not been defined. Mercury has been reported to cause pancytope nia in man. In vitro studies have shown that mi cromolar concentrations of both inorganic and organic mercury inhibited colony formation in primary cul ture of mouse bone marrow (46). Addition of a toxic chemical to marrow cultures may suggest myelo toxicity but further confirmation by in vivo studies are necessary. We have used a mouse model to investigate immunotoxicity. Our general experimental design employs the female B6C3F1 hybrid mouse exposed to chemicals at sublethal concentrations and frac tions thereof. A series of cell and humoral mediated immunoassays, in addition to clonal marrow assays are performed. This review deals principally with the latter by describing the effects of a variety of environmental pollutants on the pleuripotent he mopoietic stem cell (CFU-S) and the granulocytemacrophage progenitor (CFU-GM). Tetrachlorodibenzo-p-dioxin (TCDD) is an envi ronmental pollutant that is ubiquitous, persists in the environment, and causes varying degrees of , . ,marrow hypocellularity in all species of animals studied to date 47). Mice were exposed to 0 1 0 5.0 or 15 jug/kg body weight of TCDD pre- and postnatally by maternal dosing (48). As shown Table l,both 5 and 15 p,g/kg dosage groups showed a significant reduction in marrow cellularity, CFU-S and CFU-GM. Hematology profiles and blood smears revealed a normocytic anemia in these mice (48); bone narrow toxicity was correlated with depressed immunologic and host resistance responses. Polybrominated biphenyls (PBB) were accidently introduced in cattle and chicken feed in Michigan and resulted in widespread human exposure 49). Mice were exposed pre/postnatally by maternal dos ing at 0, 1, 3 and lOmgkg maternal body weight (49). As shown in Table 1, there was no significant decrease in marrow cellularity or CFU-GM. The CFU-S in male mice at the highest dose was 80% of controls but the difference was not significant (50). At the 1 mgkg level, the CFU-GM numbers were significantlyenhanced. In general the study showed little or no marrow alterations and also only mini mal alterations in immunological function tests. Benzo(a)pyrene, a constituent of petroleum com pounds and derivatives, has long been recognized as a potent mutagen and carcinogen. Mice were exposed to 50, 200 or 400 mgkg benzo(a)pyrene by subcutaneous injection for 10 doses over a 14-day period. Bone marrow cellularity showed a significant increase but there was a significant and dose re sponsive reduction in both marrow CFU-S and CFU-GM (Table 1).These mice also showed a vari- Table 1 The effect of environmental chemicals on bone marrow. Chemical Dose, mgkg TCDD PBB B(a)P DES IND 0.001 0.005 0.015 1 3 10 50 200 400 0.2 2 8 1 2 4 `Percent of controls, 7 mice/group. bP < 0.05 vs. controls. 'P < 0.01 vs. controls. Cell or colony numbers, % of controls Narrow cellularity Pleuripotent stem cells (CFU-S) Granulocyte-macrophage progenitors (CFU-GM) 100* ND 121 83b 68b 90 61' 37c 2L1 91 ND 125b 95 ND 111 86 ND 108 115 80 85 105 80c 82b 120b a1 a? 84 97 16b 74 88 65c 68 59* S' 112 108 118 88 ND 122 96 81 120 132 Environmental Health Perspectfaas ety of immunological defects, primarily of humoral immunity (51). Diethylstilbestrol (DES) was formerly prescribed as a synthetic estrogen and also was used to fatten cattle, Human residues accumulated from ingestion of dairy and meat products have been found. Mice were exposed by subcutaneous injection to 0.2, 2 and 8 mgkg body weight for 5 consecutive days (52). There was a dramatic decrease in marrow cellularity, CFU-S and CFU-GM (Table 1). These animals also exhibited highly enhanced sensitivity to lastada monocytogenes and other generalized immunotoxic responses (55), Indomethacin (IND) is a potent anti-inflammatory agent that appears to act by inhibiting prostaglan din synthetase. Mice were exposed to 0, 1,2 and 4 mgkg by subcutaneous injection for six consecutive days. In general there tended to be some enhance ment of CFU-GM with no significant alterations in marrow cellularity or CFU-S (Table 1). These ani mals showed enhanced resistance to Listeria monocytogenes (54). _ Mice exposed to orthophenylphenol or tris(2,3dichloropropyl) phosphate exhibited no alteration in bone marrow cellularity or CFU-GM numbers (55). Conclusions Clonal marrow assays have greatly aided our understanding of hematopoiesis and factors control ling the production of blood cells. These assays are currently finding widespread use in cancer chemo therapy centers as tools to understand and to moni tor myelotoxicity following antineoplastic therapy. We have shown that some environmental pollutants likewise have effects on marrow cellularity and progenitor cells and that these effects are readily detected by clonalmarrow assays. It would appear that wider application of these techniques can help define myelotoxicity resulting from chemical expo sure. Anemia iscommonlyreported in longterm chronic toxicity tests. This would suggestthat myelotoxicity assessment panels should also include assays for erythroid progenitors. Erythropoiesis can also be assessed by in vitro clonal assays or by S9Fe uptake ** vivo. Recently it has been shown that hemopoietic have a limited renewal capacity and that to these cells can induce a permanent de crease in their proliferative capacity (20). 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Laboratory studies on polybrominatedbiphenyl-in^sj immune alterations following low-levelchronic or prefposjgtoi exposure. Int. J. Immunopharmac. 2: 69-80(1980). Jp 51. Dean, J. H., Luster, M. I., and Boorman, G. A. Eflwtrf Environmental Health Persi [a)pyrene on immunologic, bone marrow and host re parameters. Manuscript in preparation. , G. A., Luster, M. I., Dean, J. H., and Wilson, E. The effect ofadult exposure to diethylstilbestrol in the on macrophage function and numbers. J. ReticuloS" (100n) . J. H ,Luster, M. I., Boorman, G. A., Luebke, R. W., Lauer, L. D. Effect* of adult exposure to diethylstilbeson tumor susceptibilityand host resistance parameters. J, Beticuloendothel. Soc. 2$: 571-584 (1980). 54. Boorman, G. A.. Luster, M. I., Dean, J. H.,and Luebke, R. W. Effect of indomethacin on the bone marrow and immune system in the mow . In preparation. 55. Luster, M. I., Dean, J. H., Boorman, G. A., Archer, D. L., Lauer, L. D., Lawson, L. D., Moore, J. A., and Wilson, R. E. The immunotoxicity of orthophenylphenol, Tris (2,3- dichlompmpyl phosphate) and cyclophosphamide following subchronic exposure in mice. Toxicol. Appl. Pharmacol., in press.