Document g21ZJamrvYE7nVxB9zYzj4mba

0 CIIT ACTIVITIES Vol. 8,No. 1 January, 1988 Studies on the Mechanisms of Chemically Induced Leukemia/Lymphoma Richard D. Irons, Department of Cellular and Molecular Toxicology Leukemias associated with potential exposure to chemicals or viruses are a major concern in modernsociety. The mechanismof leukemogenesisin rodents is poorly understood, making risk assessment of human exposure difficult. Studies at CllT are attempting to elucidate the respective roles of chemical exposure and retroviruses in leukemogenesis in order to evaluate the appropriateness of animal models for extrapolating risk to man and to provide information necessary to identify human populations at risk. Few diseases command the respect may behave like solid tumors or Bone Marrow Damage, Myelodysplastic that is associated with leukemia or alternatively spread via the blood or Syndrome, and Leukemogenesis lymphoma. Despitetherapeutic advances lymph. Because a significant number of in recent years, most notably with leukemias or lymphomas share a A predominant feature linking leukemia Hodgkin's disease, many leukemias and common origin in the blood-formingcells in man to experimental models of the lymphomas remain among the most of the bone marrow and also share other disease is the association between aggressive cancers known, with survival biological characteristics, they are often leukemia and damage to the bone after diagnosis often measured in months classified together. On occasion, marrow. Although great efforts have been rather than years. Secondary leukemias, determining whether a given disease made in recent years to refinetherapeutic those occurring as a result of radiation, should be classified as a lymphoma or a modalities, leukemias remain the most chemotherapy or exposure to benzene, leukemia is difficult or even arbitrary. frequent neoplasms arising secondary to carry a particularly dismal prognosis. chemotherapy and/or radiation Leukemias are neoplasms of the Stem Cells and Blood Formation employed in the treatment of cancers, hematopoietic system (blood and blood- transplantation rejection or immune ,forming organs) that usually are All the cells of the blood, including complex diseases. Similarly, experimen- disseminated via the blood. Lymphomas erythrocytes (red blood cells), leukocytes tal models of leukemogenesis-be they are neoplasms of the immune system that (white blood cells) and platelets, share a spontaneous or induced through common heritage in a single type of cell exposure to drugs, chemicals, radiation, Ithat normally resides in the bone marrow. or viruses-invariably involve the .This "pluripotential stem cell" (PSC) is hematopoietic (blood producing) stem The Author capable of either replicating itself or cells that reside in the bone marrow and Dr. Irons joined the staff of CllT in January of 1977 and is currently a senior scientist in the Department of Cellular and Molecular Toxicology. He is a recognizedauthorityon blood and bone marrow toxicity and is author of numerousscientificarticles and book chapters on the subject. He holds membership in several scientific and professional organizations, is past chairmanof the NIH Toxicology Study Section, and is a member of the EPA Health Effects Review Panel and the Toxicology Information Program Committee of the National Academy of Sciences. giving rise to any of the various cell types found in the peripheralblood and immune system. To appreciate this feat, one should consider that in the normal adult the frequency of PSC in the bone marrow is between 1 in 1000and 1 in 10,000 cells, yet the bone marrow replaces over 10,000,000,000 (10") blood cells every day and is capable of increasing this output as much as sixfold in order to meet increasing demands. The process through which this occurs involves a complex interaction of growth factors, accessory cells and feedback mechanisms that regulate the differentiation and replication of blood precursor cells and in turn the production ultimately give rise to the cells of the blood and immune system. It is now recognized clinically that acute leukemia is often preceded monthsor even years by functional abnormalitiesof the blood and bone marrow, and that bone marrow suppression, aplastic anemia and myeloid leukemia represent a continuum or spectrum of blood dyscrasias (abnormalities) rather than separate or unrelated entities. Since these functional changes \involve abnormalities in the growth of blood precursorcells. they have been collectively termed myelodysplastic syndrome (MDS) (Galton, 1986). MDS describes a variable but' well- of mature blood cells (Fig. 1). (Continued on page 3) ISSN 8755-4259 PubIications Activities is published monthly by the Chemical Industry Institute of Toxicology. All correspondence should be directed to Willanna Griffin, Information Services, Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, North Carolina 27709. Telephone (919) 5412070. Robert A. Neal, Ph.D., President James E. Gibson, Ph.D. Vice President and Director oi Research -Donald A. Hart, Ed. D., Vice President Administration and Secretary Cattley, R. C., and Popp. J. A. (1987). Barrow, C. S. (1987).One-year inhalation Effect of the peroxisome proliferator toxicity study of chlorine in rhesus hepatocarcinogen WY-14,643 on foci of monkeys (Macaca mulatta). Fundam. altered hepatocytes i n d u c e d b y Appl. Toxicol. 9,557-572. diethylnitrosamine and phenobarbital in Short, 8. G., Burnett, V. L., Cox, M. G., rats. Proceedings of the 38th Annual Bus, J. S., and Swenberg, J. A. (1987). Meeting of the American College of Site-specific renal cytotoxicity and cell Veterinary Pathologists, p . 131. proliferation in male rats exposed to [Abstract]. petroleum hydrocarbons. Lab. Invest. Irons, R. D.,Stillman, W. S., and Cloyd, 57(5), 564-577. M. W. (1987). Selective activation of Starr, T. B. (1987). The delivered/ad- endogenous ecotropic retrovirus in ministered dose relationship and its hematopoietic tissues of B6C3F1 mice impact on formaldehyde risk estimates. during the preleukem.2 phase of 1,3- I n : P h a r m a c o k i n e t i c s i n R i s k butadiene exposure. Virology 161, 457- Asssessment [Drinking Water and Health, 462. vol. 81. National Research Council, Klonne, D.R., Ulrich, C. E., Riley, M.G., National Academy Press, Washington, Hamm, T. E., Jr., Morgan, K. T., and D.C., 327-333. SUPPORTING COMPANIES Air Products and Chemicals, Inc. Allied-Signal, Inc. Amoco Chemical Company ARC0 Chemical Company BP America, Inc. A Subsidiary of The Standard Oil Company Bristol-Myers Company The Dow Chemical Company E. I. du Pont de Nemours & Co., Inc. Eastman Kodak Company Ethyl Corporation Exxon Chemical Company GAF Corporation General Electric Company Hoechst Celanese Corporation W. R. Grace (L Co. IC1 Americas Inc. The Lubrizol Corporation Mobil Research and Development Corporation Monsanto Company Occidental Chemical Corporation Olin Corporation Phillips 66 Company Polysar Incorporated PPG Industries, Inc. The Procter & Gamble Company Quantum Chemical Corporation Rohm and Haas Company Sandoz Crop Protection Corporation Texaco Inc. Union Carbide Corporation Presentations and Interactions Butteworth, B. E. Chairman, Session Proliferation Subgroup, Risk Sciences on Cellular and Biological Aspects of Institute, Washington, DC, November 4, Promotion and Progression, Conference 1987. on Dermal Carcinogenesis: Research Popp, J. A. "Mechanisms of Directions for Human Relevance, Austin, Hepatocarcinogenicity of Peroxisome TX, December 1 4 , 1987. Proliferating Chemicals," Department of Conway, J. G. "Role of Oxidative Pathology, Colorado State University, Ft. Damage in Hepatocarcinogenicity by Collins, CO, November 19, 1987. Peroxisome Proliferating Compounds," Popp, J. A. Appointed to AIDS Advisory Alcon Laboratories, Ft. Worth, TX, Committee, National Institute of November 17, 1987. Environmental Health Sciences, Dean, J. H. Group Discussion: December 1987. Repeated-Dose Toxicity Testing, Target Popp, J. A. "Peroxisomes and Organ Toxicity; and Group Discussion: H epa t o c a r c in o g e n i c it y, " Has k e lI Neurobehavioral Toxicology, Behavioral Laboratory for Toxicology & Industrial Teratology. Sterling-Winthrop Research Medicine, E. I.du Pont de Nemours(L Co., Institute, Rensselaer, NY, December 3, Inc., Wilmington, DE, December 3, 1987. 1987. Popp, J. A. "Rodent Liver Tumors and Heck, H. d'A. ExpertWorking Group on Their Relationship to Human Risk Formaldehyde, International Program on Assessment," Meeting of the CllT Board Chemical Safety, World Health of Directors, Research Triangle Park, NC, Organization. Hannover, Federal December 9, 1987. Republic of Germany, November 9-13. Popp, J. A. "Comparative and 1987. Quantitative Aspects of Rat Liver Monticello, T. M., Wong, K.L., Everitt,J. Medium-Term Assays," Meeting on I., Popp. .J. A., and Morgan, K. T. Development of New Medium-Term "Histopathology of the Nasal Epithelium Bioassays for Carcinogenesis, U. S.- of Rhesus Monkeys Exposed to Japan Cooperative Research Program, Formaldehyde Gas," Thirty- eighth Honolulu, December 15, 1987. Annual Meeting of the American Welsch, F. Appointed to Developmental Association of Veterinary Pathologists, Toxicity Profile Organizing Committee, Monterey, CA, November 12, 1987. Health Effects Research Laboratory, U.S. Neptun, D. A 1988 Chairman, North Environmental Protection Agency, Carolina Section of the American December 1987. Association for Clinical Chemistry, Welsch, F. Appointed to Reproductive December 1987.b and Developmental Toxicology Program Popp, J. A. "Further Studies on Review Subcommittee, N a t i o n a l P e r o x i s o m e P r o l i f e r a t i o n a n d Toxicology Program, Board of Scientific Hepatocarc inogenic ity ," Peroxisome Counselors, December 1987. @ChemicalIndustry lnsfifufe of Toxicology (CllT), 1988. All Rights Reserved. 2 I LebkemWLymphoma (from page 1) recognized set of blood disorders that have high propensity for terminating in acute myeloid leukemia (AML). Previously defined diseases that are included within MDS are aplastic or refractory anemia and preleukemia (Galton, 1986). Frequent signs include: anemia, leukopenia (a decrease in white blood cells), and thrombocytopenia (decrease in platelets), accompanied by increased cellularity of the bone marrow. The rate of transformationof MDSto AML varies from study to study but is about 30/o in "spontaneous" MDS and between 50 and 70% in MDS occurring secondary to chemotherapy or benzene exposure. The relationship between MDS and benzene leukemogenesis appears to be exceptionally strong, with MDS preceding the onset of leukemia in virtually every case for which data is available (Bagby, 1986). However, determining whether MDS precedes every case of benzene-induced leukemia remains an impossibility. It should be pointed out that secondary MDS itself is considered a neoplastic process and carries a grave prognosis,with a mortality of between 30 and 40% for those cases that do not progress on to AML. Evidence exists to indicate the great majority of cases of MDS and most leukemias are the result of abnormalities originating within a single stem cell, i.e., they are clonal in origin (McCulloch eta/., 1982). Studies utilizing cytogenetic or biochemical markers to identify the origins of abnormal cell populations have revealed that such clonal abnormalities may be present for years prior to the clinical recognition of leukemia. Thus a paradigm hasemergedthat implicatesthe stem cell in a multistep process that ultimately leads to the presentation of leukemia. Elucidation of the molecular events that precede leukemogenesis is a goal yet to beachieved.However, it seems reasonable to assume that at least one fundamental lesion must involve structural changes at the level of the genome (Le.,a mutation) of the stem cell. Certainly, the propensity of alkylating agents to cause leukemia, the prevalence of chromosomal abnormalities in leukemias and their clonal origin provide a rational basisfor thisconcept. However, since many leukemias are characterized by what appear to be normal blood cells that are abnormally regulated with respect to growth and maturation, it has been postulated that the genetic information may be normal but regulation defective. In this regard it is pr.?bablynot insignificant that certain normal cells of the hematoporetic and immune systems (e.g., lymphocytes) have characteristics usually ascribed to cancer cells, such as the ability to replicate, invade other (Continued on page 4) SELF-RENEWING STEM CELL COMPARTMENT Schematic Diagram of Hematopoiesis I COMMITTED I MORPHOLOGICALLY IDENTIFIABLE I STEM CELL COMPARTMENT I -PRECURSOR CELLS I MATURE BLOOD I CELLS I I PRE+LYMPmX;YTr - POCYCHROMAX)PHILIC ERITHROBLIST I T TE -I I I I 1 BLVMPHaME IRETICULOCYTE CFU-E PROERYTHRO- m I L I C BL*sI ERYTHROBL*ST ORTHOCHROMATIC ERYTHROBUST +--I .-I CFUGEMM(M1X) ___f CFWS I I BUST I I +@-I I @-m II MYELOBLAST PROMYELOWE MYELOCYTE METAMYELO- BClND II - CVTE GMNULOCYTE I ___) 1 II I I uoNocITE/ MACROPH*GE Fig. 1. The production of blood cells involves the progressive maturationand replication of precursor cells in the bone marrow. This process begins with the pluripotential stem cell (PSC), which has limited replicative abillty but is capable of self-renewal. Alternatively, it can differentiate to produce cells committed to any of the different blood cell pathways. Differentiationalong a committed pathway Is accompanied by a progressive i n c r a m in replication such that the initial division of a PSC is amplifieda billion-fold prior to the appearance of mature blood cells. Various assays have been described that measure stem cells by detennining their abillty to form colonies (/.e., colony-fonning units-CFU). Several of these have been found to measure stem cdls at owdapping st8ges in the commitment process. Assays that measure stem cells with the capability of self-renewal include CFU-S (spleen) and CFU-mix (or granulocyte, erythrocyte, megakaryocyte, macrophage). Assays that measure multipotential and committed stem-cell populations Include: BFU-E (burst forming unit-erythrocyte), CFU-E (erythrocyte), CFU-M (megakaryocyte), and CFU-GM (granulocyte-macrophage). 3 I Leubemia/Cymphoma (from page 3) tissues, kill cells, etc., but that the number and activity of these cells are normally held in check by highly effective and redundant control mechanisms. Since clonal abnormalities can be present for long periods of time prior to the onset of frank leukemia, there is considerable likelihood that both hypotheses are correct. It may be that structural damage to the DNA results in a heritable defect that conveys a proliferative advantage on a target stem cell, presumably via altered regulation of any number of potential cellular "oncogenes," but ultimate expresion of the malignant phenotype requires intervening influencesthat favor or select for abnormal gene expression but are not necessarily themselves genetic events. Viruses and Leukemia In John Steinbeck's tragedy Of Mice and Men, the mouse standsasa symbol of man's inability to understand and control his destiny. To almost everyone involved in the study of leukemogenesis, the analogy is an undeniablyapt one. Murine models of leukemogenesis present perhaps the best opportunity to unlock the. mysteries of this disease. Nevertheless, the investigatorattempting to use these animal models is confronted with a puzzle of Gordian proportion. One of the culprits implicated in this dilemma is a family of retroviruses, commonly referred to as murine leukemia viruses (MuLV). Retroviruses are RNA viruses that commandeer the normal machinery of a cell to produce a complementary piece of DNA which is then inserted into the nuclear DNAof thecell. Once this proviral DNA sequence'is integrated into the cell, it becomes a permanent part of its genome, encoding for the production of retrovirus (Fig. 2). In a great number of species, including man, retroviruses have been implicated in many diseases of the blood and immune system, including leukemia and acquired immune deficiency syndrome (AIDS) (Table 1). The first human retrovirus, T-cell lymphotrophic virus (HTLV-I) was discovered in 1978 (Poiesz et a/., 1980; Hinuma et a/., 1981; Popovic et a/., 1982). It causes adult T-cell leukemiaandto date appears to be restricted to southeastern Japan, the Caribbean, certain regions in Italy, the southern United States, South America and Africa. HTLV-I is an infectious retrovirusthat is passed horizontally, Le., from one person to another. As such it is an exogenous retrovirus, capable of being transmitted from one blood cell to another, but is not transmitted vertically from one generation to another through the germ cells. If the target of a retrovirushappens to be an ovarian or testicular germ cell, the encoded proviral sequence will be 4 inherited as a "normal" gene by all further Infection of a Target Cell by a Retrovirus progeny. Retroviruses inherited in such a manner are called "endogenous" to TARGET CELL distinguish them from exogenous or infectious retroviruses. None of the known infectious human retroviruses (e.g., HTLV-I, -11, HIV) are known to target germ cells and thus to be transmitted vertically. Nevertheless, virtually all mammals, including man, possess some DNA sequences encoding for retroviral elements. In some species, including man, endogenous sequences are rareand there is yet no evidence to indicate that they areexpressed. However,it isa legacy of the great majority of strains of laboratory mouse to possess numerous Fig. 2. A retrovirustargetsandenterscells genes encoding for retroviruses. This by interacting with receptors On the cell includes the CD1, C57BL/6 and C ~ Hplasma membrane. Upon entering the strains as well as the ~ 6 ~ h3ybr~id 1cell, the retrovirus releases its RNA into frequently used in carcinogenicity the CYtOPlaSm. A viral enzyme, reverse studies. Certain of these endogenous transcriptase, catalyzes the synthesis Of retroviruses are known to produce a high DNA COmPlementaW to the Viral RNA, frequency of "spontaneous" leukemias in which is then integrated into the cell's specific strains of mice,e.g. AKR and C58 nuclear DNA. Thus the information (Hartleyetal., 1977;Chattopadhyayetal., encoded in the viral RNA is passed on to 1982), are implicated in certain strain- any daughter cells produced through specific models of radiation-induced subsequent division of the cell. leukemogenesis (C57BU6, C3H and RF) (Gross, 1959; Leiberman and Kaplan: 1959; Kaplan, 1967; Upton et a/.. 1958: and Leukemogenesis Haran-Ghera. 1976) .and appear to influence the incidence of 1,3-butadiene- A number Of and drugs are induced leukemia in the ~ 6 ~ 3 ~ 1known to cause bone marrow toxicity in (Irons et a/., 1988). In spontaneous man or experimental animals, and it is leukemias of the mouse, the role of therefore not surprising that they have retrovirus isdefinitive (Cloydetal., 1980), been imp1icated in experimental yet it must be said that the specific leukemogenesis. These include benzene, mechanisms or role played by virus inany butadiene, ethylene oxide, and a wide of the other models of leukemogenesis variety Of alkylating agents. To remains obscure. And in at least one benzene is the only occupational hazard model, 3 ~ m e ~ ~ y l ~ ~ o l a n ~ ~ r ~denfieni~teilynli~nkuecdteo a~cute leukemia in man. leukemias i n the RF mouse, no Although seemingly unrelated, all of convincing role for retrovirus has yet these agents share One Or more been established (Goodenow and Lilly, characteristicsincommon: They all either characteristic e t a'" common to al1l 9n8a5tu)r'allAy occurring mammalian retroviruses, both distribute to the bone marrow in appreciableconcentrationsorgive riseto hreaalf-clitvivees mientabbioolloitgeiscawl ithsyrsetleamtivse. lyThloensge de(eixr.oeggc.,etlMynoutruLaVsn)s(,efio.sgrmt.h, HiantTgtLhQVee-yIn)deooorrne6ondtdoconogcneotnagoienunase,.tmoestuarbvoivliec intermediates, in turn, are able transit to the bone marrow and in their coding sequences. Therefore, any are capable Of further bioactivation Or proposals to account for the roleof these dtiisrseucte.inteFrarocmtiontwheith cpeellrsspinetchteivteargoetf viruses in leukemogenesis must invoke alternative explanations. (Continued on page 5) TABLE 1 RETROVIRUSES CAUSINGPATHOLOGY IN NATURALHOSTS vlrus Dlroawr 1. Fellne LeukemiaVirus (FeLV) Leukemia, anemia, immunosuppression 2. Bovine Leukemia Virus (BLV) Lymphoma 3. Mouse Leukemia Virus (MuL Leukemia, lymphoma 4. Simian L mphoma Virus (SLJ :5. Human?Cell LeukemiaVirus I (HTLV-I -8. Human T-cell LeukemiaVirus II HTLV-b) -7. Equine Infectlous Anemia Virus (LIAV) Lymphoma Adult T-cell leukemia Histiocytidreticulumcell leukemia Anemia 8. Visna Virus Neurologicaland pulmonary disease in sheep 9. Human ImmunodeficiencyVirus AIDS (LAV/HTLV-III/HIV) Leukemia/Lyrnphorna (from Page 4) implicated in spontaneous and radiation- correlates with leukemogenesis (Irons et understanding the role of chemical exposure in experimental leukemogenesis and its significance with respect to human health, two of these compounds, induced models of leukemogenesis. Ecotropic MuLV proviral sequences are incomplete in the NIH Swiss and no viruses are found following BDexposure (Irons et a/., 1987b).We therefore felt that a/.,1986a,b; I987 a,b,c). Although its role in leukemogenesis is presently unknown, retrovirus activation has been shown to precede spontaneous leukemia development in other mouse models benzene and 1,3-butadiene, represent an a comparison of the B6C3F1 and NIH involving virus (e.g., AKR mice). interesting dichotomy. Benzene is one of Swiss strains represented a reasonable Furthermore, treatment of B6C3F1 mice few industrial or environmental agents for first approach to determining the with 5-azacytidine, a prototype activator which there is sufficient data to establish influence of this class of retroviruses on of mouse retroviruses (Niwa and its leukemogenicity in man. Nevertheless, leukemogenesis associated with BO Sugahara, 1981), also results in the history of researchinto its mechanism exposure. IeukemiaAymphoma. Thus, current of action has been one repeatedly We were able to confirm that thymic evidence suggests that the measurement hampered by the lack of an appropriate lymphoma is the major cause of death in of retrovirus activation may be a useful animal model for benzene-induced B6C3F1 mice chronically exposed to BD screening tool for identifying potential leukemogenesis. Butadiene, on the other for one year and that the specific tumor leukemogens in the B6C3F1 mouse hand, is a potent leukemogen in the type is a T-cell lymphoma (Irons et a/., model. B6C3F1 mouse; however, understanding 1986~)W. e also determined that the bone Preliminary characterization of BD- thesignificanceof this model with respect marrow is a major target of BD toxicity induced thymic lymphomas reveals them to occupational exposure to 1.3- (Irons et a/., 1986a.b; Leiderman et a/., to be uniformlyof T-cell origin, toexpress butadiene poses a formidable challenge. 1986).The bone marrow has consistently varying amounts of retroviral gene been found to be a target in animal and products, and to be clonally derived human leukemias and the megaloblastic (Irons et a/., 1987a). Tentatively, these Butadiene anemia observed in the B6C3F1 mouse tumors appear to be arrested at a following BD exposure has fea!ures particularly late stage in the lymphocyte Although not acutely toxic to humans similar to MDS. In the B6C3F1 mouse, differentiation pathway and to proliferate or animals, 1,3-butadiene has been traditionally used in carcinogenesis via growth factor-dependent pathways demonstrated to be a carcinogen in mice bioassays, the incidence of spontaneous employed by normal T lymphocytes. Inall and rats (Huff et a/., 1985; Owen, 1981). leukemia is low and endogenous of these aspects, they are curiously Previous studies have revealed an r e t r o v i r u s ( M u L V ) expression is analogous to HTLV-I associated T-cell extremely high incidence of thymic suppressed. Nevertheless, results of our leukemias in man. Further analysis of lymphoma/leukemia in the B6C3F1 studies suggest that MuLV plays a role in these tumors may enable us to evaluate mouse but not in ratschronicallyexposed BD- induced leukemia. First, we the potential interactions of virus and to butadiene, and in 1983 a research discovered an unusual selective chemical with cellular macromolecules program was inaugurated at CllT to study activation of a class of MuLV (ecotropic) involved in the transformation process. the mechanisms of 1,3-butadiene- induced lymphoma/leukemia (Irons, 1985).At that time, the major goal was to ascertain the relevance of using the B6C3F1 mouse as a model for the extrapolation of risk of leukemia to man. that occurs during the preleukemic phase of BDexposure (Irons, et a/., 1987a,b). Preliminary studies indicate that the mechanism of viral activation probably involves de novo increases in virus production in individual cells rather than Using this model system, it may be possible to simultaneously evaluate the respective contributions of genetic structural alterations in proviral sequences and proto-oncogenes as well as structural or functional alterations of Although this is still a major objective, the alterations in the ability of the mice to surface molecules that regulate cell results of studies in our laboratory and restrict virus movement. In addition, we growth and behavior at the plasma others have led us to realize that it is also observed a marked difference between membrane. important that we determine if the mouse the incidence of lymphoma observed in While these findings are provocative can be used as a model for virus B6C3F1 and NIH Swiss mice exposed to and provide a presumptive indicationthat activation, and if chemicals can interact BD (50% and lo%, respectively) (Irons et MuLV are involved in BD-induced with viruses in producing neoplasiaeither a/., 1988). Significantly, toxicity to the lymphoma/leukemia. it presently cannot in the mouse or man. bone marrow was qualitatively and be ruled out that other genes may Because of the frequent association of quantitatively identical between the two contribute to differences in susceptibility endogenous retrovirus with spontaneous strains despite the difference in leukemia between these two strains. Nevertheless, lymphomas in the mouse, the finding of incidence. In fact, of all the parameters it appears likely that the number of viable increased thymic lymphoma in the (both cytogenetic and hematologic) scenarios for the role of retrovirus can, for B6C3F1 mouse at least suggested the evaluated in these studies, activation of p o s s i b i l i t y of m o u s e r e t r o v i r a l retrovirus is the only endpoint that (Continued on page 6) involvement. Understanding the particular role or roles that a retrovirus might play in this process is a daunting TABLE 2 task; nevertheless,in practical terms, the number of possibilities can be reduced to a relatively small number. Several Accounting for Mechanisms of BD-induced Leukemogenesis in the Mouse: Alternative Scenarios alternative scenarios were initially evaluated (Table 2). Our initial approach was to (1) characterize the pathogenesis 1. BD exposure resulted in altered mouse retroviruses that were by themselves of BD-leukemogenesis in the B6C3F1 leukemogenicbut that BD alone was not. mouse, including the molecular alterations associated with BD toxicity and tumor development; and (2) compare the incidence of leukemia in B6C3F1and NIH Swiss mice chronically exposed to 2. Both activated retrovirusesand BD were independently leukemogenic. 3. Mouse retroviruses were necessary but not leukemogenic by themselves (Le., cocarcinogenic). 4. Mouse retroviruses were activated by BD but played no role in leukemogenesis. BD (1250ppm for one year). A particular 5. Mouse retroviruses were not activated by BD and played no role in leu- class of MuLV, ecotropic MuLV, has been kemogenesis. 5 Leukemia/Lymphoma (from page 5) acute myelogenous leukemia (AML) been disappointing. However, as a (Vigliani and Forni, 1975; Infante et a/., consequence o f recent studies -all practical purposes, be reducedto two: either activated retroviruses and BD are independently leukemogenic, or MuLV is- 1977). Benzene-induced AML's are predominant Iy myelob last ic (FAB c I ass if icat io n M 1) , aI th0 ugh a conducted by Dr. Eugene Cronkite at Brookhaven National Laboratories and results obtained in experimentsat CIIT, it co-carcinogenic, i.e., a significant particularly strong association has been would now appear that these difficulties influence but not by itself leukemogenic established between benzene exposure have been largely overcome. in this chemical leukemogenesis model and the erythroleukemic variant of AML Early studies at CllT revealed that (Table 2). These alternatives can be (FAB classification-M6) (Galavotti and toxicity of benzene or its metabolites to tested experimentally. In addition, it Roisi. 1950; Aksov et a/.. 1976). Further e o n e marrow cells followed a Pattern becomes necessary to determine how studies have suggested .an association exhibited by certain cancer chemother- valid are mouse models of retrovirus with lymphomaas well (Aksoyetal., 1974; apeutic agents that are toxic to cells only activation as predictors of the potential of Vianna and Polan, 1979; Bernard, 1942). at certain stages in their growth cycle chemicals to alter the biology of From the results of these and other (Ironset a/., 1979).The proportion of cells retroviruses, such as HTLV-I or HIV, in studies one can safely conclude that affected following exposure to such a human cells. Although little is known occupational exposure to benzene is cycle-specific agent is governed by the about the mechanism of HTLV-I associated with an increased risk of number of cells in a sensitive phasc transformation, evidence strongly hematologic neoplasms. regardless of the dose of the compound suggests that environmental factors Despite the weight of evidence administered (Ironsand Horan, 1978). For influence latency in HTLV-l-associated implicating benzene as a leukemogen in such agents, the regimen or temporal neoplasms, and chemically induced man, there is no clear understanding of pattern of exposure may be equally as alterations in HTLV-I antigen expression the pathogenesis of the disease or the significant as the actual exposure has been reported in human cells in vitro. quantita ti ve relat ionsh ip between concentration. By examining the potential of butadiene benzene exposure and leukemogenesis. While investigating the effects of or its metabolites to alter the biology of Although chronic exposure to 100 ppm concentration and duration of exposure HTLV-I in human blood cells in culture, benzene is known to result in bone on benzene-induced bone marrow we hope to clarify the significance of the marrow damage and an increased suppression in mice, Dr. Cronkite and his mouse retrovirus model to man and incidence of leukemia in man, the co-workersadopted an exposure regimen epidemiologic findings that may suggest significance of exposure to low that deviated significantly from the a population at potential risk. In addition c o n c e n t r a t i o n s o f b e n z e n e i s traditional lifetime exposure protocols to qualitative analyses of the effects of controversial. A prevailing sentiment p r e v i o u s l y used t o s t u d y t h e chemicals on viruses in human and among clinical hematologists is that leukemogenicity of benzene. They mouse cells, it may be possible to blood dyscrasias indicative of bone exposed mice for 16 weeks to 300 ppm quantitate relative differences in species marrow damage frequently, if not benzene and held them for lifetime sensitivity at a cellular level. This would invariably, precede the development of observation. Two models of benzene- be of considerable value in extrapolating secondary AML. However, it presently induced leukemiahavebeendescribedby in vivo animal data to man. cannot be concluded that frank bone Cronkite using this regimen: thymic m a r r o w damage i s a n absolute lymphoma/leukemia in the C57BU6 Benzene prerequisite in every case. Thus, the mouse, a mouse known to carry MuLV evidence linking blood dyscrasias or and found to have a high incidenceof this Since late in the last century, leukemia with exposure to benzene at tumor type following radiation exposure; occupational exposure to benzene has lower concentrations i n man is and myelogenous leukemia in the been associated with bone marrow controversial, and there is little CBA/Ca mouse, which has a very low toxicity (Santesson, 1897). The most agreement among clinicians, epidemi- spontaneous incidence of AML but is frequently reported effects have been ologists, and researchers as to the known to develop a high incidence of cytopenias (a decrease in one or more of significance of exposure to benzene at AML after exposure to radiation the different types of circulating blood low concentrations (Infante et a/., 1977; (Cronkite, 1987). leukocytes or white cells), anemia and Harris, 1977). Itis not known,forexample, Most recently, in experiments again pancytopenia or aplastic anemia (a whether bone marrow suppression, intended to characterize the regimen- severe and usually irreversible bone frequently seen at higher exposure dose dependence of benzene bone marrow suppression culminating in the concentrations, is a requirement for the marrow toxicity, we have obtained inability to effectively replace any of the evolution of AML, implying a threshold preliminary results that confirm and cellular elements of the blood) b e l o w w h i c h b e n z e n e i s n o t extend the use of the C57BU6 mouseas a (Goldwater, 1941). The first report of leukemogenic, or alternatively, whether model for benzene leukemia. In these leukemia associated with benzene there is no effective threshold for experiments, micewere exposed to 100or exposure appeared in 1928 (Delore and benzene. This debate is not likely to be 300 ppm, either 3 or 6 days/week for 12 Borgomano, 1928), yet benzene was not r e s o l v e d w i t h o u t a n i n c r e a s e d weeks and held for observation. At universally recognized as a human understanding of the mechanisms of approximately 6 months, MDS was leukemogen u n t i l the mid-70's. leukemogenesis. encountered in animals in each of the Recognition appears to have awaited two Historically, such studies have been exposure groups. Included i n the independent developments: (1) Although hampered by the lack of an appropriate spectrum of hematopoietic lesions scattered individualcases appearedin the animal model for benzene-induced encountered were not only Tcell literature, it remainedfor Aksoy et a/. and leukemogenesis. To varying degrees, IymphomaAeukemia but myelogenous Vigliani and Saita to provide the first investigators have been successful in leukemia as well (Cathro et a/., 1988). organized studies of benzeneexposure in demonstrating temporary or reversible With these modelswe are now afforded an the workplace; and (2) this has coincided bone marrow toxicity following benzene opportunity to investigate systematically with relatively recent recognition that exposure in rats and mice (Snyder et a/., potential mechanisms of benzenetoxicity MDS and AML represent a continuum or 1978; Snyder et a/., 1982a,b; Goldstein et to the bone marrow and leukemogenesis. spectrum of blood dyscrasias rather than a/., 1982;Cronkiteeta/., 1984; Cronkiteet The history of research in chemical separate or unrelated entities. a/., 1985; Green e t a / . , 1981). leukemogenesis suggests that numerous The principal neoplastic disease now Nevertheless, previous attempts to associated with benzene exposure is produce a useful leukemia model have (Continued on page 7) 6 3 .Leukemia/Lymphoma (from page 6) factors, including retrovirus background, the regimen of chemical exposure,and its duration may be important considerations in carcinogenesis that have so far received little attention. Understanding the relationship between chemical toxicity, retroviral genes and leukemogenesis will aid in providing an informed evaluation of both the appropriateness of the mouseasamodel for extrapolating risk of leukemogenesis to man and the relative importance of chronic high versus low level or transient hematotoxicity and leukemogenesis.Am. J. Ind. Med. 7, 447-456. Delore, P., and Borgomano, C. (1928). Leucemieaigue au coursde /'intoxication benzenique. Sur I'origine toxique de certaines leucemies aigues et leurs relationsavec les anemiesgraves. J. Med. Lyon 9, 227-233. Galavotti, R., and Roisi, F. M. (1950). Erytholeukemic myelosis in benzene poisoning. Br. J. Indus. Med. 7, 79-81. Galton, D. A. G. (1986). The myelodysplastic syndromes. Scand. J. Haematol. 36, 11-20. Goldstein, B. D., Snyder, C. A., Laskin, model for the study of mechanisms of chemically induced murine leukemia/ lymphoma. CllT Activities 5(3), 1,4-5. Irons, R. D., Cathro, H. P.. Stillman, W. S., Steinhagen, W. H., and Shah, R. S. (1988). Susceptibility to 1,&butadiene leukemogenesis correlates with endogenous ecotropic retroviral background in the mouse. Toxicologist 8(1), 2. [Abstract No. 71. Irons, R. D., Heck, H. d'A., Moore, B. J., and Muirhead, K. (1979). Effects of short term benzene administration on bone marrow cell cycle kinetics in the rat. Toxicol. Appl. Pharmacol. 51, 399-409. chemical exposures with respect to S.,Bromberg, I., Albert, R. E., and Nelson, Irons, R. D., and Horan, P. K. (1978). human health. Such information is N. (1982). Myelogenous leukemia in Application of flow cytometry for the critically important for the development rodents inhaling benzene. TOX.Lett. 13, e v a l u a t i o n of m y e l o t o x i c i t y i n of sound regulatory and industrial 169-173. experimentaI animals. Toxicol. Appl. exposure policies. Goldwater, L. J. (1941). Disturbances in Pharmacol. 45,253. the blood following exposure to benzol.J. frons, R. D., Oshimina, M., and Barrett, References Lab. Clin. Med. 26, 957-973. J. C. (1987~)C. hromosomal aberrations Goodenow, M. W., and Lilly, F. (1984). in mouse bone marrow cells following in Aksoy, M., Erdem, S.,Dincol, K., Kepy, Expression of differentiation and murine vitro exposure to 1,3-butadiene. T., and Dincol, G. (1974). Chronic leukemia virusantigensin cellsof primary Carcinogenesis 8, 1171-1174. exposure to benzene as a possible tumors and cell lines derived from Irons, R. D., Smith, C. N., Stillman, W. contributing etiologic factor in Hodgkin's chemically induced lymphomas of RF/J S., Shah, R. S., Steinhagen, W. H., and disease. Blut 38, 293-298. mice. Proc. Natl. Acad. Sci. 81,7612-7616. Leiderman, L. J. (1986a). Macrocytic- Aksoy, M., Erdem, S., and Dincol, K. Green, J. D., Carroll, A. S., LoBue, J., megaloblastic anemia in male B6C3F1 (1976). Types of leukemia in chronic Goldstein, B. D. and Albert, R. E. (1981). mice following chronic exposure to 1 , s benzene poisoning. A study in thirty-four Acute and chronic dose/response effect butadiene. Toxicol. Appl. Pharmacol. 83, patients. Acta Hematolog. 55,65-72. of benzene inhalation on the peripheral 95-100. Bagby, G. C., Jr. (1986).The conceptof bloodl bone marrow and spleen cells Of Irons, R. D., Smith, C. N., Stillman, W. preleukemia: Clinical and laboratory CD-1 male mice. Toxicol. A p p l . S., Shah, R. S., Steinhagen, W. H., and studies. CRC Crit. Rev. Oncol./Hematol. ~ h ~ m a C O5l9., 204-214. Leiderman, L.J. (1986b). Macrocytic- 4: 203-220. Gross, L. (1959). Serial cell-free megoloblastic anemia in male NIH swiss Bernard, J. (1942). La lymphocytose Passage of a radiation activated mouse mice following repeated exposure to 1 , s benzenique. Sangre 15, 501-505. leukemia agent. Proc. SOC. Exp. Biol. butadiene. Toxicol. Appl. Pharmacol. 85, s.,Cathro, H. P., Stillman, W. Med. 100, 102-107. 450-455. Steinhagen, W. H.,and Irons, R. D. (7988). Haran-Ghera, N. (1976). Pathways in Irons, R. D., Stillman, W. S., and Cloyd, Short-term inhalation exposure to murine radiation leukemogenesis- M. W. (1987b). Selective activation of benezene produces myelodysplastic coleukemogenesis. In: Biology of syndrome and leukemiain c57BL/6 mice. Radiation CafCinOgeneSiS, J. M. YUmaS, TOXiCOlOgiSt 8(1), 70. [Abstract No.2771. R. w. Tennant, and J. D. Regan, eds., Chattopadhyay, S. K., Cloyd, H. w., Raven press, New York, 245-260. Linemeyer. D. L.. Lander, M. R., Rands, E., Harris, L. (1977). Testimony before and LOWY,D. R. (1982). Cellular origin and roleof mink cell focus-forming (MCF) viruses in murine thymic lymphomas. Nature 295, 25-31. Chinsky, J., Goodenow, M., Jackson, M., Lilly, F., Leinwand, L., and Childs, G. Occupational Safety and Health Administration* U.S. Department of Labor, August 8. Hartley, J. W., Wolford, N. K., Old, L. J., and Rowe, W. P. (1977). A new class of tnUrine leukemia virus associated wtih (1985). Comparison of endogenous development of spontaneous lymphmurine leukemia virus p r o v i r a l omas. Proc. Natl. Acad. Sci. USA 74,789- endogenous ecotropic retrovirus in hematopoietic tissues of B6C3F1 mice during the preleukemic phase of 1,3- butadiene exposure. Virology 161, 457462. Irons, R. D., Stillman, W. S., Shah, R. S., and Cloyd, M. W. (1987a). Selective activation of endogenous ecotropic retrovirus in tissues of B6C3F1 mice during the preleukemia phase of 1,& butadiene exposure. J. Cell. Biochem. 11A, 204. Irons, R. D.,Stillman, W. S., Shah, R.S., organization and RNA expression in 3- methyfcholanthrene-induced and spontaneous thymic lymphomas in RF and AKR mice. J. Virol. 53, 94-99. Cloyd, M. w., Hartley, J. w.,and Rowe, w. p. (1980). Lymphomagenicity of recombinant mink cell focus-inducing murine leukemia viruses. J. Exp. Med. 151, 542-552. Cronkite, E. P. (1987). Chemical leukemogenesis: Benzene as a model. Semin. Hematol. 24, 2-11. Cronkite, E. P., Bullis, J. E., ~noueT, ., and Drew, R. T. (1984). Benzene inhalation produces leukemia in mice. Toxicol. Appl. Pharmacol. 75, 358-361. 792. Hinuma, Y., Nagata, K., Misoka. M., Nakai, M., Matsumoto,T., Kinoshita, K. I., Shirakawa, S., and Miyoshi, I . (1981). Adult T-cell leukemia: Antigen in an ATL cell line and detection of antibodiesto the antigen in human Sera. PrOC. N d . Acad. sei. USA 78, 6476-6480. Huff, J. E., Melnick, R. L., Solleveld, H. A., Haseman, J. K., Powers,M.,and Miller, R.A. (1985). Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1miceafter 60 weeks of inhalation. Science 227,548- 549. Infante, P. F., Rinsky, R. A., Wagoner, J. K., and Young, R. J. (1977). Leukemia in Morris, M. S., and Higuchi, M. (1986~). Phenotypic characterization of 1,s butadiene-induced thymic lymphoma in male B6C3F1 mice. Toxicologist 6. 21. Kaplan, H. S. (1967). On the natural history of the murine leukemias: Presidential address. Cancer Res. 27, 1325-1340. Leiberman, M., and Kaplan. H. S. (1959). Leukemogenic activity of filtrates from radiation-inducedlymphoid tumors of mice. Science 130, 387-388. Leiderman, L. J., Stillman, W. S., Shah, R. S., Steinhagen, W. H., and Irons, R. D. (1986). Altered hematopoietic stem cell development in male B6C3F1 mice Cronkite, E.P., Drew, R. T., Inoue, T., benzene workers. Lancet I / , 76-78. and Bullis, J. E. (1985). Benzene Irons, R. D. (1985). 13Butadiene: A (Continued on page 8) 7 Leukemia/Lymphoma (from page 7) following exposure to 1,3-butadiene. Exp. Molec. Pathol. 44, 50-56. McCulloch, E. A., Smith, L. J., and Minden, M. D. (1982). Normal and malignant haemopoietic clones in m w . Cancer Surv. 1, 280-298. Niwa, O., and Sugahara. T. (1981). 5Azacytidine induction of mouse endogenous type-C virus and suppression of DNA methylation. Proc. Natl. Acad. Sci. USA 78, 6290-6294. Owen, P. E. (1981). The toxicity and carcinogenicity of butadiene gas administered to rats by inhalation for approximately 24 months. Final Report. [Vol. 11. Hazleton Laboratories, Europe Ltd., Harrogate, England. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D.,and Gallo, R. C. (1980). Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77, 74157419, Popovic, M., Lalyanaraman, V. S., Sarngadharan, M. G., Robert-Guroff, M., Nakao, Y., Reitz, M. S., Miyoshi, Y., Ito,Y., Minowada. J., and Gallo. R. C. (1982).The virus of Japanese adult T-cell leukemia in a member of the human T-cell leukemia virus group. Nature 300, 63-66. Santesson, C, G. (1897). Chronic poisoning with coal tar benzene: Four deaths. Clinical and pathologicalanatomical observations of several colleagues and illustrating animal experiments. Arch. Hyg. (Munich) 31, 336-376. Snyder, C. A., Goldstein, B. D., and Sellakumar, M. A. (1978).Hematotoxicity of inhaled benzene to Sprague-Dawley Rats and AKR mice at 300 ppm. J. Toxicol. Environ. Health 4, 605-618. Snyder, C. A,, Goldstein, 8. D., Sellakumar, A. R., Bromberg, I., Laskin, S., and Albert, R. (1982a).The inhalation toxicology of benzene: Incidence of hematopoietic neoplasms and hematotoxicity in AKR/J and C57BL/6 mice. Toxicol. Appl. Pharmacol. 54, 323331. Snyder, C. A., Goldstein, B., Sellakumar, A., Bromberg. I., Laskin, S. and Albert R. (1982b).Toxicity of chronic benzene inhalation: CD-1 mice exposed to 300 ppm. Bull. Environ. Contam. Toxicol. 29, 386391. Upton, A. C., Wolff, F. F., Furth, J., and Kimbail, A. W. (1958). A comparison of induction of myeloid and lymphoid leukemias in X-irradiated RF mice. Cancer Res. 18. 842-848. Vigliani, E. C., and Forni, A. (1975). Benzene and leukemia. Environ. Res. 11, 122-127. Vigliani, E. C., and Saita, G. (1964). Benzene and leukemia. N. Engl. J. Med. 172, 872-876. Vianna. N. J., and Polan, A. (1979). Lymphomas and occupational benzene exposure. Lancet I , 1394-1395. 8 Service Awards Presented Twenty-six CIIT empioyees were recognized for five and ten years' service at the institute's Christmas party on December 12. Reachlng the ten year mark were (seated, left to right): Dr. Richard J. Levine, Epidemiology; Charles A. Howard, Building Services; Dr. Henry d'A. Heck, Biochemical Toxicology and Pathobiology; Dr. Byron E. Butteworth, Genetic Toxicology; Betsy Gross Bermudez, Experimental Pathology and Toxicology; and Diane J. Abemethy, Genetic Toxicology. Standing, left to right: Edna A. Mangum, Administration; Delorlse H. Williams, Experimental Pathology and Toxicology; Corrie N. Smith, Experimental Pathology and Toxicology; Charles M. Overton, Purchasing; and Dr. Douglas E. Rickert, Biochemical Toxicology and Pathobiology. Not pictured is Donald F. Deyo, Biochemical Toxicology and Pathobiology. Recognized for five years' sewlce were (seated, left to rlght): Gary Trent Jones, Experimental Pathology and Toxicology; Betty A. Honeycutt, Genetic Toxicology; Linda K. Garvey, Experimental Pathology and Toxicology; Karen M. Dold, Cellular and Molecular Toxicology; Dr. Jack H. Dean, Cellular and MolecularToxicology; and Kaye S. Nagy, Purchasing. Standing: R. Dwight McFarland, Building Seruices; Carl U. Parkinson, Jr., Experimental Pathology and Toxicology; Dr. Thomas R. Skopek, Genetic Toxicology; Dr. Frank Welsch, Cellular and Molecular Toxicology; and Donald 8. Stedman, Cellular and Molecular Toxicology. Five-year employees not pictured are Dr. William F. Greenlee, Cellular and MolecularToxicology; Lloyd D. Lauer, Cellular and Molecular Toxicology: and Mary S. Morris, Experimental Pathology and Toxicology. 0