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UNWERSITY OF OTTAWA Faculty of Medicine Faculty of Health Sciences Benzene State of the Science Workshop December 16 & 17,1998 Institute ofPopulation Health Faculty of Medicine and Faculty of Health Sciences BP-00011551 Agenda- December 16, 1998 Benzene~orksbop University of Ottawa Institute of Population Health 8:30am Welcome Daniel Krewski Rapporteur 8:45am Overview ofthe Benzene Problem Robert Snyder Benzene Metabolism and Genotoxicity, Bob Sonawane, Chair 9:15am -9:35am 9:45am Benzene Metabolism David Ross Paul M. Schlosser, Discussant Open Discussion lO:OOam Break 10:15 am Benzene-induced Genotoxicity John Whysner 10:35 am David A. Eastmond, Discussant II 10:45 am Open Discussion ll:OOam Implications for Risk Assessment Daniel Krewski, Rapporteur Toncity & Leukemogenesis, Patrick Beatty, Chair 11:15 am 11:35 am !1:40am Value ofTransgenic models John E. French Robert Snyder, Discussant Discussion !2:00pm Lunch 1:10pm Hematotexicity Modeling Patrick Beatty Melvin Andersen 1:30pm 1:50pm2:00pm Secondary Leukemogenesis Richard A. Larson Armand Keating, Discussant Discussion 2:15pm 2:35pm 2:45pm Hematotoxicity & Leukemogenesis Azra Raza Debra L. Laskin, Discussant Discussion 3:00 pm Break 3:15pm 3:35pm 3:55pm 4:05pm Mechanism..ofLeukemogenesis Richard D:-lrons George F. .Kalf Martyn T. Smith, Discussant aiscussion 4:25pm 4:40pm 5:00pm Implications for Risk Assessment Conclude Daniel Krewski, Rapporteur Discussion 7:00pm Dinner; Dinner speaker: Ron Worton, University of Ottawa BP-00011552 Agenda- December 17, 1998 Benzene Workshop University of Ottawa Institute of Population Health Benzene Induced Leukemia: Human Studies, Carol Henry, Chair 8:30am 8:50am 9:10am 9:30am 9:50am NCI/Chinese Epidemiology Study Insights from Petroleum Studies Biomarkers of Leukemia Risk HEI Biomarker Research in China New Initiatives for Epidemiologic Research Richard B. Hayes/Martha Linet Robert Schnatter Martyn T. Smith Martha E. Richmond Otto Wong 10:10 am Break 10:30 am Discussion Panel Jack Siemiatycki Pierre Band David Bayliss Bob Sonawane 11:1-5 am Open Discussion 11:55 am Implications for Risk Assessment Daniel Krewski, Rapporteur 12:10 pm Lunch Future Research to Improve Risk Assessment & Risk Management, Bernie Goldstein, Chair 1:00pm U.S. EPA ~erspective BruceRodan 1:20pm Health Canada Perspective Bette Meek 1:40pm European Union Perspective E. Dinant Kroese 2:00pm California EPA Persp-ective Lauren Zeise 2:20pm Petroleum Industry Perspective Patrick Beatty Geoffi-ey Granville 2:40pm Good Science for Good Decisions; The NIEHS Perspective Christopher Schoenwalder 3:00pm Open Discussion 3:45pm Concluding Remarks Daniel Krewski, Rapporteur 4:00pm Conclude BP-00011553 1998 Benzene State of the Science Workshop University of Ottawa.-lnstitute for Population Health Observations for Presenters Thank you for agreeing to participate in this important workshop. Our goal for the two days we will be _together is to establish a road-map for future benzene research. As you are already aware, the purposes of this workshop include: reviewing the current state ofknowledge about benzene leukemogenesis identifying data gaps and areas where further research would- contribute meaningfully to scientific understanding ofthe mechanism and extent ofbenzene's-human hematotoxicity and leukemogenesis understanding the implications ofthe benzene database for risk assessment and for the regulatory context in which the science is used. The following questions are intended to assist you in focusing your thinking about what should be presented at the workshop in Ottawa, and about the direction your discussion should pursue. Our purpose is to focus your thoughts on the present state ofthe science and the future direction research should be directed. We also want to help you maintain the broader perspective ofthe application ofthis research to the risk assessment process. Benzene Metabolism and Genotoxicity - B. Sonawane, Charr Benzene Metabolism How can estimates ofthe 1eukemogenic risk of benzene be improved by knowledge of: 1.) The identity ofthe -leukemogenic metabolite(s) of benzene. 2.) Target tissue levels ofbenzene-metabolites. 3.) The enzymology ofbenzene metabolism. 4.) The role of metabolites transported to the bone marrow from other or~ans vs. in situ formation. In the above, presenters might consider the iJlications for dose-response, biomarkers of exposure, possible-mechanisms ofbematotoxicity and genotoxicity, and inter-individual variatian in hematotoxic and leukemogenic response. In what experimental systems can useful information be developed, e.g. experimental animal studies, human in vitro, studies of exposed human populations'! Benzene-Induced Genotoxicitv Although the weight ofthe experimental evidence strongly suggests that benzene does not induce point I mutations, there are data from multiple techniques which, suggest that benzene derived mate!:ial binds to genetic material.- Numerous studies have linked benzene exposure with chromosomal aberrations and certain chromosomal rearrangements are associated with AML. How important to benzene risk assessment is distinguishing between direct DNA binding of benzene and chromosomal rearrangements BP-00011554 0 1998 Benzene State of the Seience-Workshop -as critical to the leukemogenic process? What are the implications for the dose response characteristics of benzene-induced leukemia, and how- might this question be answered? To what extent can specific chromosomal rearrangements be identified as specific to leukemia secondaryto chemical exposures as opposed to de novo leukemia? What are the implications for risk assessment ofvarious proposed mechanisms ofbenzene-induced clastogenicity, e.g. DNA- protein crosslinks,-interference with mitotic apparaws, topoisomerase inhibition. Toxicity & Leukemogenesis- P. Beatty, Chair Some ofthe core questions that may be addressed in this-Session include: 1.) What are the mechanisms by which reactive metabolites ofbenzene influence bone marrow function leading to aplastic anemia? 2.) What are the mechanisms by which reactive metabolites ofbenzene influence genotoxicity, chromosome damage, and transformation to a neoplastic cell? 3 .) Does benzene-induced leukemia develop through a series of steps similar to the classic carcinogenic model, i.e. initiation, promotion, progression? Hematotoxicity and Leukemogenesis At least one multi-factorial modetof benzene inauced leukemia has been proposed which includes an interplay of both genotoxic and non-genotoxic mechanisms. Based upon a model ofthis basic Conn: 1.) By what mechanisms oould hematotoxicity play a role in the induction of leukemia, e.g., effects on apoptosis or alteration ofproliferation or differentiation of critical cell types? 2.) Is hematotoxicity (as determined by what endpoint) an obligatory part ofthe leukemogenic process? What are the implications for risk assessment ofthe involvement ofhematotoxicity in the leukemogenic process, e.g., shape ofthe dose-response, importance ofpeak versus cumulative exposures for risk? Hematotoxicity Modeling This presentation is expected to summarize the modeling workshop held the day before the State ofthe Science Wolkshop. lnJ:he summarization, a focus should be on__how well the toxicodynamic model proposed by Cox describes the development of benzene toxicity or leukemogenesis, and how it could be used in the risk assessment process. Secondary Leukemogenesis A number of othe~ chemicals are known to cause leukemia, primarily AML, secondary to exposure. Some ofthese compounds require metabolic activation while others are direct acting; some compounds are linked to specific biochemical mechanisms such as inhibition oftopoisomerases while the leukemogenic mechanism of others is not readily apparent. Can the actions ofbenzene on the bone marrow,_such as the development of myelodysplasia, lead to the characterization of benzene as a "secondary leukemogen?" Ifso, where does benzene fit into this spectrum-Of secondary leukemogens, and can this infonnation provide useful guidance for risk assessment BP-00011555 ------------------ 199_8 Benzene State of the Science Workshop Page 3. of benzene induced leukemia, e.g. leukemia cell type for quantitative potency calculations, relative roles ofgenotoxic vs. non-genot.oxic mechanisms & dose-response implications? Value ofTransgenic Models The history ofresearcll into benzene leukemogenesis has been plagued by the lack of an animal model for this endpoint. Initial reports indicate that oral dosing of benzene to a transgenic mouse strain. Tg.AC. containing an activated H-ras oncogene. results in induction ofgranulocytic leukemia. What additional information must be developed in order to validate this mouse strain as a model fur benzene-induced leukemia in humans? What questions. raised in earlier sections ofthis document, may be experimentally accessible with this model? Mechanism ofLeukemogenesis The biological mechanism of benzene-induced leukemogenesis has-been suggested to be similar the model for colon cancer, a multi-component process with the-involvement of both genetic and non-genetic mechanisms. Does the state ofthe science currently support such a model in concept? Ifnot. what additional type(s) of information would confirm or disprove the model? Elucidating the exact structure ofa multi-component model in a complex biological system such as the hematopoietic system is, at best, a complex problem, and complete knowledge may be beyond the capabilities .ofcurrent technology. Considering the implications f-or benzenes risk assessment of such a multi-component model, in what detail does the--model have to be understood-to provide useful guidance for risk assessment? For instance: 1.) Is it critical to issues such as dose response to know the temporal sequence ofthe genetic-and nongenetic components? 2.) To what extent is knowledge ofthe exact identity ofthe components of1:he mechanism necessary in order identify relevant leukemia cell types for epidemiology studies? 3.) Is it eno.ugb to detennine that oncogene activation--ls-the genetic event in leukemia induction or is .the identity ofthe oncogene(s) involved necessary? Benzene Induced Leukemia: Human Studies -C. Henry., Chair Epidemiologic Research Exposure assessment is a common area of weakness in most occupational and environmental epidemiology studies. Problems often encountered include the need to estimate or model exposures from limited monitoring data, and the presence ofother, potentially confounding. exposures that cannot be adequately controlled for. Recognizing exposure assessment as a fundamental issue for epidemiologic research into the leukemogenic effect ofbenzene, presenters may wish to consider. "1.) what are the limitations of exposure data from the studies being considered, and-ofthe studies that have been used to date in regulatory risk assessment? 2.) Aie there additional_data on exposure which could be accessed and analyzed to enable more accurate quarifification of exposure-response for existing studies, or for future planned studies? 3.) Row might such information be accessed and analyzed? BP-00011556  1998 Benzene State of the Science Workshop Page 4. 4.) What is the minimum dataset for quantification ofexposure in epidemiologic studies needed in order to accurately estimate dose-response. and hence to use the study in a risk assessment? 5.) Are there data available which would permit meaningful additional analysis to shed light on risks associated with peak versus cumulative exposure? In considering potential confounding exposures and other co-factors, presenters may consider addressing issues such as: 1.) Are there data which would permit systematic investigation of substances other than benzene to which subjects in their studies may have been exposed, or may potentially be exposed. and how could these data be brought into play? 2.) How should exposures to other chemicals be considered in understanding the risk associated with exposure to benzene? 3.} Would it be possible or worthwhile to systematically investigate socioeconomic and ethnic differences which might contribute to variatiOns in risk between North American and Chinese populations? 4.) Would additional analysis ofconfounders, such as smoking or familial history ofcancer, contribute meaningfully to our knowledge base about risks in petroleum workers associated with exposure to benzene? Other issues presenters may wish to consider in formulating their remarks include: 1.} What are the plausible scientific reasons for the variations in findings from the different studies? How plausible is it that these variations are due only to the variations of sensitivity ofthe different investigations? 2.) Is it feasible to investigate incidence/mortality due to acute myelogenous leukemia alone in further analyses ofbenzene-ilxposed cohorts? What is the importance ofconsidering specific cell-types and is it feasible to do this using the epidemiologic method? Biomarkers Biomarkers-present a potential opportunity to enhance the understanding of human health risks from benzene exposure. Presenters may wish to consider 1.) What is the technical feasibility of using biomarkers for benzene research? 2.) Are there ethical considerations in the use ofbiomarkers? 3.) How would benzene biomarkers relate to other biomarkers that have been identified? 4.) Are there correlations between biomarkers of exposure and biomarkers ofeffects, as they relate to benzene and leukemia? 5.) How could biomarkers be incorporated into epidemiologic research, and what potential do they have for strengthening the understanding ofassociations between benzene and health effects? 6.) Is there a way ofattributing biomarkers-to specific sources of exposures? 7.) How should biomarkers be utilized in the risk assessment process, and what4"!eigbt should be ascribed to studies that use them? - - - ..---. BP-00011557 Abstracts Benzene State of the Science Workshop December 16-&17, 1998 BP-00011558 Overview of the toxicology of benzene. Robert Snyder, Ph.D., Professor and Chairman, Department of Pharmacology and Toxicology, Rutgers, the State University ofNew Jersey and Associate Director, Environmental and Occupational Health Sciences Institute, Piscataway, NJ, USA. Santesson (1897) and Selling (1910), reported that chronic exposure to benzene in the workplace resulted in marked decreases in circulating blood cells and subsequent fatality. An overview of benzene-induced bone marrow depression can be derived from industrial studies where large groups ofworkers were exposed and various stages in the severity of the disease could be identified among the worker population, e.g., Helmer (1944), Greenburg et al. (1939), Hamilton-Patterson and Browning (1944), Savilahti (1956). Aksoy et al. (1971). Chronic ex-posure to low concentrations of benzene may produce reversible decreases in blood cell numbers. However, higher chronic exposures lead to the onset of irreversible bone marrow depression which is characterized by anemia, leucocytopenia, with emphasis on lymphocytopenia, and/or thrombocytopenia.:-Decreases in all three cell types is defmed as pancytopenia, which in the case of benzene toxicity results from severely damaged bone marrow, and the disease is termed aplastic anemia. The recognition that benzene was a leukemogen was established on the basis of studies among exposed worker populations by in Italy by Vigliani and Forni (1976), in Turkey by Aksoy and Erdem (1978), in the United States by Infante et al. (1977) and in China by-Yin et al. (1987). Decreases in circulating blood cells may also be observed in the presence of a dysplastic marrow. Myelodysplastic syndrome (MDS) is a preleukemic state characterized by abnormal marrow architecture, inadequate hematopoiesis, and many cells demonstrating chromosome damage. MDS is a clonal disease (Janssen et al., 1989) which may result in response to chemotherapy by alkylating agents (Kantarjian and Keating., 1987) or to chronic benzene exposure (Forni and Moreo, 1967,1969; van den Berghe et al., 1979). Chronic benzene toxicity leading to MDS usually proceeds to acute myelocytic leukemia (AML). Chromosome damage has been associated with benzene~induced leukemia since the observations ofPollini et at-(1964) and Forni et al. (1971). Damage to chromosomes 5 and 7 have been associated with MDS (Jacobs et al., 1986) and AML -(Golomb et al., 1982) and after benzene exposure (Sasiadek, 1992). Smith et al., (1998) have demonstrated translocations and aneusomy in chromosomes 8 and 21 after benzene exposure. Whether the result of benzene exposure is aplastic anemia or leukemia, death usually results from infections because among the effects of benzene is depression of the immune system. Immunotoxic effects of benzene have been known since the early 1900's (Snyder and Kocsis, 1975). Benzene depresses B-and T-lymphocytes as well as their mitogenic responses (Rozen and-Snyder, 1'985). Lymphocytes are decreased both in the circulation and in the bone marrow in rats and-mice ( Wierda and Irons, 1982). Mice treated with benzene by subcutaneous injection displayed fewer peritoneal macrophages, decreased phagocytic -and tumor cell cytolysis capability,.and increased hydrogen peroxide generation (Klan et al., 1990). Within the bone marrow an increased number of macrophages, enhanced chemotaxis, elevated hydrogen peroxide production, as well as increases in mononuclear phagocytes were. obsetved (MacEachern et al., 1992)~Furthermore, nitric oxide production in bone marrow macrophages, which appears to be tied to benzene-induced decreases in hematopoiesis, is increased (Laskin et al., 1995). The data suggest that impairment ofmacrophage activity is mediated by benzene BP-00011559 metabolites (Lewis et al, 1988; Manning et al., 1994). The expression of impaired immune function was demonstrated by Rosenthal and Snyder (1985) who reported that benzeneexposed mice demonstrated decreased resistance to infection by L. monocytogenes. The processes of differentiation and amplification which mark the pro~ssion from pluripotential stem cells through the various stages of maturation bas an absolute requirement for a functional hematopoietic microenvironment This system, referred to as the stromal cells of the bone marrow, includes macrophages, endothelial cells, reticular cells, fat cells, etc. These cells provide nutrients, growth factors and an environment necessary for nonnal bone marrow function. Using a technique which involves long term bone marrow cell cultures, Garnett et al. (1983) showed that stroma from benzene treated mice were ineffective in supporting the differentiation -of stem cells from untreated mice. Gaido and Weirda (1984,1985,1987) reported that hydroquinone and benzoquinone decreased the ability of stromal cells to support G!M colony formation. Stromal macrophage production of IL-l was anoinhibited by hydroquinone (Renz Kalf (1991). A hypothesis worth considering is that damage to the stroma may be the most significant factor in the decrease in circulating cells that is ultimately termed aplastic anemia Thus, benzene toxicity is a continuum of events leading from decreases in circulating blood cells, to pancytopenia and aplastic anemia or to MDS and acute non-lymphocytic leukemia. Chronic exposure is necessary, but the limits on the "dose X time" relationship are only partially understood. Because of the impairment of the immune system, death may result as a function of marrow dysplasia or aplasia; or in leukemia. Benzene was one of the prototype compounds which led to the understanding that toxicity of organs other than liver may be the result of metabolites formed in the liver , as well as, in target organs. Inlubition of benzene metabolism (Andrews et al., 1977) or partial hepatectomy (Sammet et al., 1979) protected against benzene-induced decreases in erythropoiesis. All-of the known unconjugated metabolites of benzene, with excepti-on of phenol and-1,2,4-benzenetriol have been shown to decrease erythropoiesis (Snyder and Hedli. 1996). Furthermore, the evidence indicates that the toxic effects result from interaction between benzene metabolites. Eastmond et al. (1987) reported that phenol and bydroquinone interacted to exacerbate decreases in bone marrow cellularity and Guy et al. (1990) showed that combinations of either phenol plUs hydroquinone, or phenol plus catechol, were more hematotoxic than any of the metabolites given alone. The most potent and effective combination in inhibiting erythropoiesis is hydroquinone plus muconaldehyde (Snyder et al., 1989). There appear to be several mechanisms by which,benzene metabolites impair cellular functions. For example, Irons and Neptun (1980)-suggested that hydroquinone derived from 'benzene covalently binds to tubulin. a protein essential for spindle formation in mitosis, and thereby, inhibits cell replication. Schwartz et al. (1985) demonstrated that benzene metabolites inlubit mitochondrial DNA polymerase. Kalf et al., (1996l reported that p-benzoquinone inhibits the conversion of pre-interleukeins-1 and -1 to..the active cytokine. Snyder et al. (1978) found that benzene metabolites were covalently botllld to protein in several body organs after treating mice with radiolabeled benzene and Lindstrom et al. (1998) reported on specific adducts of benzene oxide with hemoglobin and albumin in mice, rats and humans. Rushmore BP-00011560 et al.(1984) demonstrated that benzene metabolites covalently bind to mitochondrial DNA and inlubit mitochondrial RNA synthesis. Damage to nuclear DNA by__benzene metabolites has- been studied in several laboratories. Lutz and Schlatter (1977) observed benzene metabolites bound to hepatic DNA after exposing animals -to radiolabeled benzene by inhalation. Structures of benzoquinone adducts to guanine, adenine and cytosine have been proposed (Jowa et al., 1990; Pongracz et al., 1991). DNA adducts arising from benzene have been measured in HL-60 cells (Hedli et al., 1996; Levay et al., 1992; 1993). Although there have been reports of DNA adducts from benzene in vivo, the level of adduct formation is not consistent and when reported is at a very elow level. Thus, Reddy et al. (1994) failed to detect DNA adducts in benzene treated mice using the 2P]post labeling assay, whereas Creek et al. (1-997), using accelerator mass spectrometry reported observing adducts to both protein and DNA in mice given [14C]benzene in the nglkg range. An alternative hypothesis for DNA damage is oxidation of DNA by reactive oxygen species that arise as a result ofbenzene metabolites. For example, p-benzoquinone and hydroquinone increase superoxide, nitric oxide, and hydrogen peroxide in HL-60 cells activated with phorbol ester (Rao and Snyder, 1995). Boersma et al. ( 1994) suggested that hyaroquinone itself is unlikely to generate reactive oxygen species. However, Brurunark and Cadenas (1988) reported on a metabolic pathway from hydroquinone leading to the production of glutathionyl-benzenetriol, which ---<:an undergo autooxidation leading to superoxide formation. An interesting corolla:ry mechanism, proposed by G. Rao ( 1994} suggests that benzene induced DNA damage is mediated by (1) release of free iron in the bone marrow of benzene-treated animals [probably by polyphenolic metabolites of benzene], followed by (2) the chelation of iron by hydroquinone or benzenetriol to yield (3) a reactive oxygen-generating species such as superoxide, which in -tum (4) causes oxidative damage to-DNA. His suggestion that glutathionyl hydroquinone may be a key intermediate (Rao, 1996) resonates well with- the hypothesis of Brunmark and Cadenas 1988). In a radical rich system glutathionylhydroquinone may well be converted to glutathionyl-benzenetriol. In Singh et al. (1994) he suggests a-chemical-structure for the iron-benzenetriol chelate in which the iron is bound between the hydroxyl groups .at positions 1 and 2 on 1,2,4-benzenetriol with the release of two protons. Indeed, it may well be that the au-t-ooxidation of glutathionyl-benzenetriol is enhanced with an iron chelated between positions 1 and 2. A synthesis of the ideas of Rao et al. and Brurunark and Cadena.s__suggests-that the formation and autooxidation of glutathionyl,-.benzenetriol yields superoxide. The iron- benzenetriol chelate mediates the conversion of superoxide to singlet oxygen and hydroxyl radical which can hydroxylate guanine in the 8 pesition. Richter (1997) suggested that, although 8-hydroxyguanine is the most frequently observed oxidation product of DNA, any of the DNA bases may undergo hydroxylation, and potentially result in a mutational event. An alternative action which may sensitize bone marrow cells is the ability of some benzene metabolites to inhibit topoisomerase II. Chen and Eastmond (1995) and Hutt BP-00011561 and Kalf (1996) showed that p-benzoquinone was a topoisomerase II inhibitor. They suggest that compounds having similar inhibitory activity when used to treat cancers can cause subsequent delayed leukemia. In studies of the response of bone marrow cells to hydroquinone, Irons and Stillman (1996) using human CD34+ cells reported enhanced clonogenic responses to GMCSF. Hazel et al., (1996) using mouse 320.3 myeloblast cells reported initiation of differentiation by hydroquinone and benzene. which terminated at the myelocyte stage and implicated a- role for hydroquinone at the leukotriene D4 receptor (Hazel et al., 1995). HydroqQinone can also inhibit apoptosis at the myeloblast/myelocyte stage of differentiation (Hazel et a1.,1996). These effects provide a mechanism for large numbers of immature white bloo_d cells to accumulate. In the event that one of the cells entering the expanding myelocyte pool has under,gone a leukemogenic transformation at an earlier stage in maturation, it may serveas the parent of an expanding clone ofleukemia-cells. Given that benzene metabolites may -cause DNA damage leading to a mutagenic effect, which may be reflected as a translocation, or other DNA damage such as that related to inhibited topoisomerase II, the process may be termed "initiation". The effects of benzene and hydroquinone leading to expansion of a less than fully differentiated pool of myeloid cells, provide a mechanism of "promotion". Thus, one can visualize a two step process of initiation and promotion associated with benzene-induced non-lymphocytic leukemia. The-major problem that remains-in our attempts to understand the mechanism of benzene toxicity/leukemogenesis is the interplay between exposure and sensitivity. The measurement of exposure to benzene, which defines the dose, has been difficult to achieve in retrospective studies, and dose reconstructions, usually of-questionable accuracy, is required. Sensitivity refers to factors which may render an individual sensitive or resistant to benzene. An incomplete list of variable factors includes hepatic CYP 2El (Rothman et al., 1997) activity because it is the major enzyme involved in benzene hydroxylation, the activity of conjugating enzymes of which the glutathione transferases seem to be significant, and comparative activity of myeloperoxidase _(London et al., 1997) and reductase (NQOJ) (Rothman et al., 1997) in bone marrow. Attempts to -extrapolate effects at high dose to those at low dose must take these factors into account. The issues that underlie- chronic, as opposed to acute toxicity, must also be carefully explored. Whereas, in the past the product of dose times tiine was thought to be a constant, it is now recognized- that either factor may best be described by exponential functions. It is only through a thorough understanding of the mechanism of actionlhat we can appreciate the dose response relationship. BP-00011562 REFERENCES Aksoy, M., Dincol, K., Akgun, T., Erdem, S. and Dincol. G. Haematological effects of chronic benzene. Brit J. Ind. Med. 28:296-302 (-1-971). Aksoy, M. and Erdem, S. Follow-UP study on the mortality and the development of leukemia in 44 pancytopenic patients with chronic benzene exposure. Blood 52:285-292 (1978). Aksoy, M., Erdem, S. and Dincol, S. Leukemia in shoe workers chronically exposed to benzene. Blood~44:837-841 (1974). Andrews, L.S., Lee, E.W., Witmer; C.M., Kocsis, J.J._, and Snyder, R. Effects of toiuene on the metabolism, disposition, and -hemopoietic toxicity of 3H benzene. Biochem. Phannacol. 26:293-300 (197'7). Boersma, M.G., Balvers, W.G., Boeren, S., Vervoort, J. and Rietjens, I.M. NADPHcytochrome reductase catalysed redox cycling of 1,4-Benzoquinone; hampered at physiological conditions, initiated at increased pH values. Biochem. Phannacol. 47: 1949-1955 (I 994 ). Brunmark, A. and Cadenas, E. Reductive addition of glutathione to p-benzoquinone, 2hydrozy-p-benzoquinone and p-benzoquinone epoxides. 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Hedli, C.C., -Rao, N.R., Reuhl, K.R., Witmer, e.M., and Snyder, R. Effects of .benzene metabolite treatment on granulocytic differentiation and DNA adduct formation in HL-60 cells. Archives ofToxicology. 70:135-145 (1996). Helmer, K.J. Accumulated cases of chronic benzene poisoning in the rubber industry. Acat. Med. Scand. 118:254-375 (1944). Hutt, A.M., and Kalf, G.F. Inhibition of hu.man DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene. Environ. Health Perspect. Vol. 104, Suppl. 6, pp. 1265-1269 (T996). Infante, P., Rinsky, R.A., and Wagoner, J.K. Leukemia in benzene workers. Lancet 2:76-78 (1977). BP-00011564 Irons, R.D. and Neptun, D.A. Effects of principle hydroxy-metabolites of benzene on microtubule polymerization. Arch. Toxicol. 45:297-305 (1980)- Irons, R.D. and Stillman, W.S. Impact of benzene metabolites on differentiation of bone marrow progenitor cells. Environ. Health Perspect Vol. 104, Suppl. 6, pp.l247-1250 (1996). Jacobs, R.H., Combleet, M.A., Vardiman, J.W., et al. Prognostic implications of morphology and karyotype in primary myelodysplastic syndromes. Blood 67: 1765-1772 (1986). Janssen, J.W.G., Buschle, M., Layton, M. et al. Clonal analysis ofmyelodysplastic syndromes: evidence ofmultipotential stem cell origin. Blood 73:248-254 (1989). Jowa, L., Winkle, S., Witz, G., Kalf, G.F. and Snyder, R. Synthesis and characterization of Deoxyguanosine-Benzoquinone Adducts. J. Appl. Toxicol. 10:47-54 (1990). Ka1f, G.F., Renz,""J:F. and Nicolescu, R p-Benzoquinone, a reactive metabolite of benzene, prevents the processing ofpre-interleukins-1 and -I to active cytokines by inhibition of the processing enzymes, calpain, and interleukin -1 converting enzyme. Environ. Health Persp. Vol. 104, Suppl. 6, pp. 1251-1256 (1996). KantaJjian, M.H. and Keating, M.J. , Therapy related leukemia and myelodysplastic syndromes. Oncol. Clio. 14:435-443 (1978). Klan, M:J., A-dams, D.O., and Lewis, J.G. Effects of exposure to benzene in vivo on the murine mononuclear phagocyte system. Toxicol. Appl. Pharmacal. 103:198-205 (1990). Laskin, J.D., Rao, N.R. Punjabi, C.J., Laskin, D.L. and Snyder, R. Distinct actions ofbenzene and its metabolites on nitric oxide production by bone marrow leukocytes. J. Leuko. Bioi. 57:422-426 (1995). Levay, G., Pongraz, K. andBooell, W. Detection ofDNJ\. adducts in-HL-60 cells treated with I bydroquinone andp-benzoquinone by 32 P-post labeling. Carcinogenesis 12: 1181-1186 ( 1991). Lewis, J.G., Odom., B. and Adams, D.O. Toxic effects ofbenzene and benzene metabolites on mononuclear phagocytes. Toxicol Appl Pharmacal. 92 (2):246-254 (1988). Lindstrom,.A.B., Yeowell-O'Connell, K, Waidyanatha, S., McDonald, T.A., Golding, B.T., I Rappaport, S.M. Formation of-hemoglobin and albumin adducts of benzene oxide in mouse, rat, and human blood. Chern. Res. Toxicol. 11:302-310 (1998). bandon, S.J., Lehman, T.A. and Taylor, J.A. Myeloperoxidase genetic polymorphism and lung cancer risk. Cancer Res. 15:5061-5003 (1997) butz, W. and Schlatter, C. Mechanism of the carcinogenic action of benzene: irreversible binding to rat liver DNA. Chem.-Biol. Interact 18:241-245 (1917). -MacEachern, L., -snyder, R and Laskin, D. Alterations in the Morphology and--Functional Activity of Bone Marrow Phagoe-ytes Following Benzene Treatment of Mice. Toxicol. and Appl. Pharmacal 117:147-154 (1992). BP-00011565 Manning, B.W., Adams, D.O. and Lewis, J.G. Effects of benzene metabolites on receptormediated phagocytosis and cytoskeletal integrity in mouse peritoneal macrophages. T.oxicol. Appl Pharmacol. 126:214-223 (1994). Mitelman, F., Nllsson, P.G., Brandt, L. et al. Chromosome pattern, occupation, and clinical -features in patients with acute non-lymphocytic leukemia. Cancer Gene Cytogenet. 4:197-214 (1981). Pollini, G. and Colombi, R. Chromosome damage in lymphocytes during benzene hemopathy. Med. J. Lav. 55:641 (1964). Pongracz, K. and BodelL W J. Detection of 3'-hydroxy-1,N6-benztheno-2'-deoxyadenosine 3'phosphate by 32P postlabeling of-DNA reacted with p-benzoquinone. Chern. Res. Toxicol. 4:199-202 (1991). Rao, G. Glutathionyl hydroquinone: a potent pro-oxidant and a possible toxic metabolite of benzene. Toxicology. 106:49-54 (1996). Rao, N.R. and Snyder, R. Oxidative Modifications Produced in HL-60 Cells on Exposure to Benzene Metabolites. Journal Applied ToxicoL 15:403-409 (1995). Reddy, M.V., -SGhultz, S.C., Blackburn, G.R., and Mackerer, C.R Lack of DNA adduct formation in mice treated with benzene. Mutat Res. 325(4):149-155 (1994). Renz, J.F. and Kalf, G.F., A role for interleukin-1 in benzene induced hematotoxicity: Inhibition of the conversion of pre~IL-1 to mature cytokine in murine macrophages by hydroquinone and the prevention of benzene-induced hematotoxicity in mice by interleukin-1 . Blood 78:938-944 (1991). Richter, C., Free radical mediated DNA oxidation, in: Free Radical Toxicology. pp. 89-114, Wallace, K.B. (Ed.), Target Organ Toxicology Series, Hayes, A.W., Thomas, J.A. and Gardner, D.E. (Eas.), Taylor and Francis, Washington,. DC (1997). Rosenthal, G.J. and Snyder. C.A. Modulation of the immune response to Listeria monocytogenes by benzene inhalation. Toxicol. Appl. Pharmacal. 80:502-510 (1985). Rothman, N., Smith, M.T., Hayes, R.B. et al. Benzene poisoning, a risk factor for hematological -malignancy, is-:associated with the NQOl 609C-->T mutation and rapid fractional excretion of chloroxazone. Cancer Res. 15:2839-2842 (1997) Rushmore. T., Snyder, R. and-Kalf, G. Covalent binding ofbenzene and its metabolites to DNA in rabbit bone marrow mitochondria in vitro. Chern. Biol. Interactions 49: 133-154 (1984). Sammett, D., Lee, E.W., Kocsis, J.J., and Snyder, R. Partial -nepatectomy reduces both metabolism and toxicity ofbenzene. J. Toxicol. Environ. Health. 5:785-792 (1979). BP-00011566 Santesson. C.G. Uber chronische Vergiftung mit Steinkohlenteerbenzin; vier Todesfalle. Arch: Hyg. Berl. 31:336-376 (1897). Sasiadek. M. Nonrandom distribution of breakpoints in the karyotypes of workers occupationally exposed to benzene. Environ. Hlth. Perspect. 97:255-257 (1992). Savilahti, M. More than 100 cases of benzene intoxication .in a shoe factory, Observations on hematological symptoms and course ofthe process in one year. Gewerbhyg. 5:147-151 (1956). Selling, L. Benzol as a leukotoxin. Studies on degeneration regeneration of blood and hematopoietic organs, Johns Hopkins Hospital Rep. 17:83-142 (1916). Singh, V., Ahmad, S. and Rao, G.S. Prooxidant and antioxidant properties of ironhydroquinone and iron-1,2,4-benzenetriol complex. Implications for benzene toxicity. Toxicology, 89:25-33 (1994). Smith, M.T., Zhang, L., Wang, Y., et al. Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene. Cancer. Res. 58:2176-2181 (1998). Snyder, R., and Hedli, C.C. An Overview of Benzene Metabolism. Environ. Hlth. Perspect. Volume I04, Suppl. 6, pp. 1165-1171 (1996). Snyder, R., and Kocsis, J.J. Current concepts of chronic benzene toxicity. CRC Crit Revs. Toxicol. 3:265-288 (1975). Snyder, R., Lee, E., and Kocsis, J. Binding of labeled benzene metabolites to mouse liver and bone marrow. Res. Comm. Chern. Pathol. Pharmacal. 20:191-194 (1978). Snyder, R., Di.'"llitriadis, E., Guy, R., Hu, P., Cooper, K.R., Bauer, H., Witz, G. and Goldstein, B.D. Studies on the mechanism ofbenzene tm.-icity. Environ. Hhh. Perspect. 82:31-35 (1989). van den Berghe, H., Louwagie, A., Broeckaert~van-Orshoven, et al. Chromosome analysis in two unusual malignant blood disorders presumably induced by benzene. Blood 53:558-566 (1979). Vigliani, E. and Forni, A Benzene and leukemia. Environ. Res.ll:l22-127 (1976). Wierda, D. and Irons, R.D. Hydroquinone a.'ld catechoheduce. the frequency of progenitor B lymphocytes in mouse spleen and bone marrow, Immunophannacology, 4:41-54 (1982). Yin, S.N., Li, G.L., Tain, F.D., et al. Leukemia in benzene workers, a retrospective cohort study. Brit. J. Ind. Med. 44:1'24-128 (1987). BP-00011567 Metabolic Mechanisms ofBenzene Toxicity. David Ross, Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, School ofPharmacy, University ofColorado Health Sciences Center, Denver, CO, USA._ A number ofrecent reviews have been published on benzene metabolism which provide an overview of the process (1-3). The importance of benzene metabolism to toxicity is supported by inhibition of benzene toxicity by toluene, a competitive inhibitor ofbenzene metabolism (4), the reduced toxicity ofbenzene in animals which have undergone partial hepatectomy (5) and the reduction ofbenzene toxicity in CYP2E1 knockout mice (6). The potential relevance ofputative toxic metabolites for the target organ toxicity ofbenzene and their sites ofgeneration will be discussed. Metabolic mechanisms postulated as responsible for benzene toxicity include the generation of i) benzene oxide ii) open ringed metabolites such as trans trans muconaldehyde (MA) , iii) polyphenolic metabolites such as hydroquinone, catechol, 1,2,4-trihydroxybenzene and their quinone oxidation products and iv) combinations of metabolites. Benzene oxide is formed during metabolism ofbenzene in mouse, rat and human-liver microsomes (7, 8) and can be measured in the blood -- of rats after administration ofbenzene (9). Protein adducts of benzene oxide have been detected in the blood and bone marrow ofmice and rats exposed to benzene (10). In a recent study (11) hemoglobin and albumin adducts ofbenzene oxide were detected in workers exposed to high levels of benzene. MA is formed during metabolism of benzene in mouse hepatic microsomes (12), via mechanisms which have been suggested to involve singlet oxygen or hydroxyl radical mediated oxidation ofbenzene or ring opening of the hydroxy1ated oxepin derived from benzene oxide (13, 14). MA has not-been isolated in-vivo after benzene administration although trans transmuconic acid is a metabolite ofbenzene in animals andnumans (15,16). MA and its OH/CHO derivative are both bematotoxic (13) and cytotoxic (17). Distribution studies have demonstrated that a small percentage of 14C-W.A administered to mice (ip or iv) reached the bone marrow (18). Phenolic metabolites ofbenzene are major hepatic metabolites of benzene which may undergo Phase II conjugation reactions (19). Alternatively, benzene-derived phenolics may be released into the blood (20, 21) a.qd migrate to bone marrow (22). Distribution of conjugated phenolics to distal sites followed by deconjugation also represents a possible mechanism ofdelivery ofphenolic metabolites to extrahepatic target organs (23). We have been unable to detect cytochrome P4502El in human bone marrow by immunoblotting but-marrow is rich in myeloperoxidase which can convert polyphenolics into reactive quinones. Interestingly, other target organs of benzene BP-00011568 toxicity also contain significant peroxidase activity (23.24). Quinones- can either arylate biological nucleophiles or redox cycle generating reactive oxygen species and oxidative stress. Derivatives ofquinones may alsobe responsible for toxicity. Glutathione adducts of 1,4-benzoquinone, for example, are known to be redox active and have been demonstrated in the bone marrow of mice after administration ofbenzene and are hematotoxic when administered to mice (25). Polyphenolic metabolites ofbenzene are not necessarily innocuous species and may exert mutagenic (26) and deleterious effects, such as inhibition ofribonucleotide reductase (27), independent oftheir oxidation to quinones. Combinations ofbenzene metabolites have also been suggested to play a role in benzene toxicity (1, 28-30). Since many reactive metabolites may be formed during benzene metabolism, metabolic mechanisms underlying benzene toxicity may well be multifactorial and this evidence will be -summarized. Recent work has provided evidence for metabolic susceptibility factors for benzene toxicity in humans (31 ). Occupationally exposed individuals with a higher capacity for cytochrome P4502El mediated metabolism and a lack ofNAD(P)H:quinone oxidoreductase_! (NQOI, DT-diapborase), due to a polymorphism in the NQOl gene, have an increased-susceptibility to the hematot.oxic effects ofbenzene. NQOl may function to keep benzene-derived quinones-i..'l-a reduced form where they can be detoxified by phase ll senjugation reactions or by maintaininglllltioxidant levels of ubiquinone or a-tocopherol (32) Within the bone marrow both hematopoietic progenitor cells and stromal cells are potential targets of benzene toxicity. The CD34+ progenitor compartment contains all short term and long term repopulating-cells in marrow and progenitor cells are considered likely initial target cells in leukemias. CD34+ cells contain appreciable myeloperoxidase (33, 34)-and phenolic metabol-ites of benzene have been reported to induce apoptosis in hwnan marrow CD34+ cells (35). The stroma provides a supporting framework within the medullary-eavity for blood cell development and bas been implicated as a target ofthe toxicity ofbenzene metabolites. Levels of myeloperoxidase, NQO1 and glutathione have been suggested as metabolic determinants of the effects ofbenzene-derived phenolics in bone marrow stroma (36, 37). The characterization of a single benzene metabolite or a combination ofmetabolites whicb..islare responsible for the pathological effects ofbenzene has not been possible to date. This is important for the purposes ofrisk assessment and for studies designed to elucidate the pathogenesis ofbenzene-induced bone marrow damage. Evidence supporting_the role ofeither benzene epoxide, open ringed metabolites, pherrolslquinones or combinations ofthese metabolites as putative toxic metabolites ofbenzene will be discussed as will problems associated with each potential metabolic mechanism. It is I BP-00011569 essential to assess the biological plausibility of selective toxicity at the level ofthe bone marrow for each-potential mechanism. Significant data gaps in studies of benzene metabolism and areas for future work will be identified. Particularly, the use of..stably transfected cell lines and transgenic animals represent valuable techniques that can answer questions regarding the metabolic mechanisms underlying benzene toxicity. References 1) Snyder, R. and Hedli, C.C. Environ. Health Perspect. I04" (Suppl. 6) 1165-1171, 1996, 2) Ross, D. Eur, J. Hematol. 57, 111-118, 1996; 3)R.angan, U. and Snyder. R. Annals New York Acad. Sci. 837, 105-113. 1997 4) Andrews, L.S. et al., Biochem Pharmacal. 26 293-300, 1977 5)Sammett, D. etal., J. Toxicol. Environ. Health 5 785-792, 1979. 6)Valentine, J.L. et al., Toxicol. Appl. Pharmacol. 141, 205-213 1996. 7) Tunek, A. et al. Mol. Pharmacol.., 14, 920-929, 1978 8) Lovem,.M. et al., Carcinogenesis 18 1695-1700 1997 9) Lindstrom, A.B. et al., Carcinogenesis 18 1637-1641 1997 10) McDonald T.A. et al., Cancer Res. 54 4907-4914, 1994. II) Yeowell-O'Connell, K.. et al., Carcinogenesis 19 1565-1571 1998 12) Latriano, L. et al., Proc. Natl. Acad. Sci. USA 83, 8356-8360, 1986 13) Witz, G. et al. Environ. Health Persp. 104 suppl. 6, 1195-1199, 1996. 14) Bleasda1e, C. et al., Chern. Res. Toxicol. 10 1314-1318-1997 15tParke, D.V. and Williams, R.T. Biochem. J. 54 231-238 1953, 16) Fuchs, D. and von Soo A. Hoppe-Sey1ers Z. Pbysiol. Chern. 98 11-13, 1916. 17) Goon, D. et al., Chem. Bioi. Interact. 88, 37-53, 1993. l-8~-Zhang, Z. et al., Arch. Toxicol. 71 703-8 1997 19) Sabourin, P.J. eta!., Toxicol. Appl. Pharmacal. 94 128-140 1988. 20) Medinsky, M. et aL, T-GK-icology 105, 225-233 1995, 21) Hedli, C.C. et al., Toxicol. Appl. Pharmacal. 146, 60-68 1997 22) Rickert, D. et al. Toxicol. Appl. Pharmacal. 49 417-423 1979. 23) Low, L. et al., J. Am. Coli. Toxicol. 14 40-60-1995 24) Morrison, M. and Allen, P.Z. Science 152 1626-1628 1966 25) Bratton, S.B. et.aL, Chern. Res. Toxicol. 10859-865 1997 26)-Joseph, P. et al. Br. J. Cancer 78,312-320, 1998. 27) Li, Q. et al., Toxicol. Appl. Pbarmacol.l50,154-157, 1998 28)Eastmond,D:ketal., Toxicol. Appl. Pharmacal. 91 85-95 1987 29) Subrahmanyam, V.V. et al., Toxicology 62-107-116, 1990. 30) Barale, M. et al. Mutat. Res. 244, 15-20, 1990:-31) Rothman, N. et al. Cancer Res. 57,.2839-2842, 1997 32) Siegel, D. et al., Mol. Pharmacol. 52,300-305, 1997 33) Strobl H. et al., Blood 82,2069-2078, 1993: 34) Schattenberg, D. et al., BP-00011570 Mol. Pharmacol., 46,346-351 1994. 35) Moran, J. et al.,. Mol. Pharmacal. 50,610-615, 1996 36) Ross, D. et al., Env. Health Persp. Vol104, suppl6 1177-1182 199.6; 37) Trush, MA. et al., Env. Health Persp. Voll04, suppl 6 1227-1234, 1996; David Ross Ph.D., Professor and Chairman, Department of Pharmaceutical Sciences, School of Pharmacy, University ofColorado Health Sciences Center, Denver, CO 80262, USA. Tel. 303 315 6077 Fax. 303 315 0274 -1 BP-00011571 Needs for Research on Benzene Metabolism and Dosimetry Paul M. Schlosser Chemical Industry Institute of Toxicology Research Triangle Park, NC 27709, USA In the past, benzene was used extensively in the production of paints, resins, rubber, inks, and dyes. Evidence that persons exposed to high levels of benzene for -extended -periods of time suffer an increased risk of aplastic anemia and acute myelogenous leukemia (AML) (1) prompted the adoption of regulations by the U.S. government that limited occupational exposures to benzene (2). Recent reports of increased-hematotoxicity and leukemia among benzene-exposed workers in China corroborate the existing evidence that benzene causes both aplastic anemia and AML (3,4). As in pr-evious studies, AMLin the Chinese cohort was associated with constant, high-level exposures or high cumulative exposures. Benzene is found in gasoline at a relative abundance of 1% by volume and is used in the as a feedstock for the synthesis of many organic chemicals (5). Common sources_of environmental exposure include gasoline fumes, automobile exhaust, and both mainstream and side stream tobacco smoke (6). A clear concern is whether or not these ubiquitous, low-level exposure pres-ent a significant health-risk to humans for AML, and what level of exposure can be considered to be of negligible risk. From a risk-management viewpoint, the purpose in understanding and quantifying the dosimetr:y and metabolism of benzene and its metabolites would be to use this information in characterizing the exposure-dose relationship for benzene. In particular, one would like to kAow how the concentration of key metabolites in bone marrow, including phenol, l]ydroquinone, muconaldehyde, and possibly benzene oxide, rela1es to the level of benzene to which an individual is exposed. These metabolites (with the possible exception of benzene oxide, the effects of which have not been investigated) BP-00011572 appear to play an important role in benzene-induced myelotoxicity (7, 8) and likely are significant in the induction of AML. Therefore, any nonlinearity in the exposure-dose relationship for these metabolites would be expected, in turn, to lead to nonlinearity in the exposure-response relationship for benzene-induced AML. Hence, determining or quantifying the shape of these exposure-dose relationships should improve estimates of the exposure-response relationship, particularly in extrapolating from high-level exposures at which benzene-induced AML is observed to low-level exposures of current concern. There has been considerable effort to characterize the metabolism and disposition of benzene and its metabolites in rodents (e.g., 9-13} as well as some work in non-human primates (14). These-laboratory animal data are useful currently in that they can allow one to develop and validate physiologically-based pharmacokinetic (PBPK) models in animals before attem~ting to extrapolate such models to humans. Further, one could potentially use rodent PBPK models to examine the relationship between benzene dosimetry and benzene-induced effects in rodents that might be considered precursor events and/or representative of human risks for AML. However, since there is not currently a eonfirmed animal model for AML induced by inhalation exposure to benzene, such modeling efforts are of limited utility beyond validating the PBPK model-development-process. There are two notable exceptions to the completeness of the data set for rodents. The first is the Dosimetry of trans-trans-muconaldehyde (MUC). While it has been shown that exposure -to MUC reproduces many of the myelotoxic effects of benzene exposure in rodents (7), blood levels of MUC from benzene exposure have not been reported. Thus, while M8C-has been show to have the potential to play a key role in benzene-induced toxicity, ifs actual role, which depends on how much reaches -u~e bone marrow during benzene exposures, is uncertain. BP-00011573 The second metabolite which has the potential to play a key role, and for whichthere are only very limited dosimetry (;lata to date, is benzene oxide {15). In fact, not only is the dosimetry largely unknown, but the effects of benzene oxide on bone marrow have not been evaluated. Since benzene oxide is an epoxide, and is known to form hemoglobin adducts, now that it is know to achieve measurable blood lev.els one would suspect that it _plays some role in benzene-induced toxicity. Thus, both the pharmacokinetics and the pharmacodynamics of benzene oxide should be examined. There have also been some of studies on benzene dosimetry in humans (e.g., 16-18), but these have been restricted to analyses of changes in (expired} air concentration, blood levels of benzene itself, and urinary levels of metabolites, with the later being confounded by dietary levels of phenolics (19). So one of the largest unknowns is the blood-levels of benzene metabolites in humans resulting from benzene exposure. It is theoretically~ possible to estimate these levels -using physiologicallybased pharmacokinetic (PBPK) modeling, given data now available on metabolism of benzene by human liver in vitro. But the existence- of data which would allow such predictions to be checked would add considerable certainty to the process. Anoth~r unkoown is the potential presence of cyt-ochrome P450 2E1 (CYP2E1) in human bone-marrow. CYP2E1 is found in rabbit bone marrow (20), but not that of the mouse-(21 ). While the level of CYP2E1 is likely to be low in bone marrow compared to liver, the~production of small amounts of reactive metabolites by CYP2E1 oxidation in the target tissue might have as much impact as the production of larger quantities of such metabolites in the liver, since in- the later case much of the metabolites produced may not reach the bone marrow. The final area where experimental confirmation is with~regard to the theory of Medinsky and coworkers (22) that aspects -of benzene and phenol metabolism can beexplained by zonation of metabolic enzymes in the liver. Hedli eta/. (23) reported the results of comparing benzene metabolism in the isolated, perfused mouse liver, BP-00011574 -------------- specifically comparing the results of normal (orthograde) vs. reversed {retrograde) ..perfusion. The results of Hedli et al ar.e compatible with and support tbe zonation hypothesis, but this hypothesis predicts the primary effect to be on the metabolism of phenol, not benzene. In particular, since phenol is a su9strate for sulfotransferases, which are concentrated in periportal regions of the liver, while CYPE1, which would oxidize phenol to hydroquinone, is concentrated more highly in centrilobular regions. Thus with normal perfusion of phenol one would expect the primary metabolite observed to phenol sulfate as- compared to hydroquinone, while with reverse perfusion a much higher pr-oportion of hydroquinone (and it's conjugates). Experiments to test this aspect of the hypothesis have not been performed, and would be a valuable contribution in supporting or refuting it. 1. Ayres,-PA~ and Taylor, WD. (1989) In Hayes, AH-(ed.) Principles a.f"ld Methods of Toxicology 2nd Ed. Raven Pr.ess Ltd., New York, 111. 2. Wallace, L. (1990) RiskAna/,10, 59. 3. Travis, LB, Li, CY, Zhang, ZN, Li, DG, Yin, SN, Chow, WH, Li, GL, Dosemeci, M, Blot, W, Fraumeni, JF, Jr, Hayes, RB, and Linet, MS. (1994) Leuk Lymphoma, 14, 91. 4. Xia, ZL, Jin, XP, Lu, PL. Gu, XQ; LaPorte, RE, and Tajima, N. (1995) Biomed Environ Sci, 8, 30. 5. Infante, PE, Rinsky, RA, Wagoner, JK, and Young, RJ. (1977) Lancet, 2, 76. 6. Runion, HE, and Scott, LM. (1985) Am J Jnd Med, 7, 385. 7. Smith, MT. (1996) Environ.Health Perspect, 104 {Suppl. 6), 1219. 8. Barale, R, Marrazzini, A, Betti, C, Vangelistt, V, Loprieno, N, and Barrai, I. (1990)Mutat.Res, 244, 15. 9. Rickert, DE, Baker, TS, Bus, JS, Barrow, CS, and Irons, RD. (1979) Toxicol Appl Pharmacal, 49, 417. BP-00011575 10. Gilmour, S, Kalf, G, and Snyder, R. (1986) In Biological Reactive Intermediates Ill: I Mechanism of Action in Animal Models and Human Disease, Kocsis, JJ, Jollow, OJ, Witmer, CM, Nelson, JO, and Snyder, R (eds.), Plenum Press, NY, 223. 11. Sabourin, PJ, Bechtold, WE, Griffith, WC, Birnbaum, LS, Lucier, G, and Henderson, RF. (1989) Toxicol Appl Pharmacol, 99, 421. 12 Schlosser,-PM, Bond, JA, and Medinsky, MA. (1993) Carcinogenesis, 14,2477. 13. Mathews, JM, Etheridge, AS, Matthews, HB. (1998) Toxicol Sci, 44, 14. 14. Sabourin. PJ, Muggenberg, BA, Couch, Rc-, Lefler, D, Lucier, G, Birnbaum, S, and Henderson, R. (1992) Toxicol Appl Pharmacal, 114, 277. 15. Lindstrom,_ AB, Yeoweii-O'Connell, K, Waidyanatha, S, Golding, BT, Tornero- Velez, R, and Rappaport, SM. (1997)Carcinogenesis, 1.8, 1637. 16. Inoue, 0, Seiji, K, Kasahara, M, Nakatsuka, H. Watanabe, T, Yin, SG, U, GL, Jin, C, Cai, SX, Wang, XZ, and lkeda,l~t (1986)--BrJ lnd Med, 43,692. 17. Inoue, 0, Seiji, K, Kasahara, M, Nakatsuka, H, Watanabe, T, Yin, SG, Li, GL, Cai, SX, JJn, C, and Ikeda, M. (1998) Br. J. Ind. Med., 45, 487. 18. Inoue, 0. Seiji, K, Watanabe, T, Kasahara, M, Nakatsuka, H, Yin, S, Li, G, Cai, S, Jin, C, and Ikeda, M. (1998) lnt Arch Occup Environ Health, 60, 15-. 19. Boogard, PJ, and van Sittert, NJ. (1997) Env Health Persp, 104 (Suppl. 6}, 1151. -20. Schnier, GG-.- Laethem, CL, and Koop, DR. (1989) J Pharmacal. Exp Ther, 251, 790. 21. Genter, MB, and Recio, L. (1994) FundAppl Texico/, 22,469. 22. Medinsky, MA, Kenyon, EM, Seaton,1111J, and Schlosser, PM. (1997) Env Health Persp, 104 (Suppt S), 1399. 23. Hedii, CC, Hoffmann, MJ, Ji, S, Thomas, PE, and Snyder, R. (1997) Toxicof /J.ppl Pharmacol, 146, 60. BP-00011576 BENZENE-INDUCEDG-ENOTOXICITY Jobn Whysner M.D., Ph.D. Head, Toxicology-and Risk Assessment Program American Health Foundation Valhalla, NY 10595 INTRODUCTION Benzene (BZ) is a human leukemogen that also produces neoplasia at various sites manimal bioassays. Alternative explanations for BZ-induced neoplasia based upon genotoxic effects have been proposed in the literature. This study analyzed all available studies ofDNA reactivity and genotoxicity reported in the literature for BZ and its metabolites, which involved 1400 individual test results. Based on the genotoxicity profile generated, alternative mechanisms were evaluated. DNA REACTIVITY Two types of.studies that examined DNA reactivity have been reported in the litera.tu.te: 3'lP- postlabeling, which can directly detect and identify DNA adducts, and binding ofradiolabeled BZ to isolated-DNA. DNA binding following exposures to rndiolabeled BZ has been found in both target and non-target tissues by in vivo experimental studies. In the-tissues that exhibit clear neoplastic findings, no increased level of DNA binding is apparent.compared to organs that showed no neoplastic effects. Where adducts have been clearly det-ected by 32P-postlabeling, these were not in apparent target organs for neoplasia in rodents. Conversely,_ in the tissues that exhibit clear neoplastic fmdings such as the Zymbal gland in rats, no DNA adducts have been reproducibly found. Additionally, DNA adducts have not been clearly identified by ,:zp_ postlabeling in either target or non-target tissues under dosage conditions that have provided evidence-ofneoplasia-in experimental animals. The postlabcled adduct levels were-substantially lower (about 100-fold) than those detemrlned by the DNA binding methods. The detection ofbinding by labeling methods are not necessarily an indication of covalent bindfng of chemical or its metabolite(s) to DNA since several non-DNA components could interfere in the int-erpretation .of results.. Also, these methods could detect adducts that do not have mutagenic potential. Consequently, the significance ofDNA binding studies for neoplasia are not presently interpretable. GENOTOXlCJTY snJDIES In vivo, BZ was strongly and consistentl;r positive for micronucleus formation. BZ itself was negative in the one, published, in ,itro micronucleus assay, both with and without-metabolic activation. In contrast, polyhydroxybcnzcne BZ metabol-ites, including phenol (PH), hydroquinone (HQ), catechol (CA1) and benzcnetriol (Bl), were positive in vitro in the micronucleus assay. Pata for ring-opened BZ .biotransformation products tram, trans- -l BP-00011577 ' muconaldehyde; (*MUC) and trans,trans-muconic acid (MUC) were limited. ResUlts for BZ in :' micronucleus assays were xepresentative ofthose in other assays for cytogenicity. Exposwe fn ' vivo to BZ or in ,,-itro to BZ biotransfonnation products caused ~ctural chromosomes abenations, chromosome loss (e.neugenicity), and induced strand bre'lkagc. In these other in vitro cytogenicity assays> BZ and PH were negative or positive depending-on the assay conditions, while HQ, CAT, BQ, and BT were consistently positive. BZ in vitro gave mixed positive and negative results without activation in assays for aneugenicity and clastogenicity. The positive fmdings were primarily obtained in studies employing marrow cells or leukocytes. These cell types are known to contain various enzymes that are involved in BZ myelotoxicity and genotoxicity. ln in vitro assays, the addition of induced :rat liver microsomes did not have a consistently positive or negative effect on genotoxicity endpoints. This was attributed to various factors, including absence ofenzymes critical to the true gcnotox.ic mechanisms ofBZ in vivo and, possibly, abundance ofenzymes that could detoxify -BZ biotransformation products. No specific BZ metabolite has bee:n identified to fully explain BZ genotoxicity. However7 the polyhydroxylated metabolites and their semiquinone and quinone oxidation products are clearly involved. Some mixtures ofBZ oxidative biotransfonnationproducts exhibited genotoxic; synergy in vitro. Others were additive and-others less than additive. HQ +PH+ CAT or HQ + CAT were synergistic in micronucleus assays. This finding provides evidence that specific combinations of BZ biotransformatioa products may lead to much higher levels of g.enotoxicity than either product alone. In contrast to the consistently positive data in cytogenicity assays. BZ and its biotmnsformation products, including MUC and "'MUC, were negative in assays forreversjon in Salmonella. Some positive results in assays for mammalian forn-ard mutagenicity in culture were interpreted as reflecting cytogenetic mechanisms, in the absence ofscqumce or other information necessary to conclusively determine the nature ofthe tmdcrlying event. The mammalian forward mutagenicity findings are consistCDt with a cytogenetic agent that causes chromosome breakage, loss. and/or rearrangement MECHANISMS OF GENOTOXICITY Four specific..mechanisms have been most frequently cited in the literature to explain BZ genotoxicity. as follows: 1) DNA-reactive BZ metabolites fonning adducts or cross-liDks; 2) Oxidative DNA damage leading to strand breaks and missense mutations; 3) Clastogenesis due to topoisomc:rase II inhibition; 4) Aneugenesis due to damage to components ofthe mitotic apparatus. "The gcnotoxicity profile for BZ compared to that ofother agents known to produce genotoxicity by specific mechanjsrns provided a means to differentiate between the_possible contributions of any or all ofthese mechanisms to the BZ genotoxicity findings. 2 I BP-00011578 Polyhydroxylated BZ metabolites have been shown to inhibit topoisomerase n, which p~ a double strand break allowing the passage ofa second double helix through the break. nie genotoxicity profile ofBZ matches that ofa topoisomerase II inhibitor, which is capable of producing all ofthe positive genotoxicity results through a mechanism invalving a stable BZtopoisomerase II-DNA complex. The result of forming this complex is sister chromosome exchange, non-homologous recombination. gene deletion and rearrangements. The genotoxicity profiles do not support DNA-reactivity, .genention ofreactive oxygen species, spindle poisoning, or ribonucleotide reductase inhibition as the underlying mechanism. HUMAN LEUKEMOGENESIS Studies of the genotoxicity ofBZ and its metabolites in humans and in cell derived from humans in vitro are consistent with the genotoxicity data in animals, animal-derived cells in vitro, and bacterial systems. A mechanism involving DNA reactivity is not supported by the laclfof identified DNA adduc:ts under_rclevant exposure conditions. Fmthermore, the lack of mutagenesis in bacterial tests by BZ metabolites also supports a lack ofDNA reactivity. The production ofBZ metabolites in humans is qualitatively similar to-rodents. Therefore, inhibition of-t-opoisomerase II also appears to be the best explanation ofthe genotoxicity results in humans for BZ and its metabolites. Topoisomerase ll inhibitors are known to produce leukemia in humans although the some ofthe chromosomal alterations are diffi:rmt from tbose involved in BZ-induced leukemia. Consequently, ifthe results ofgenotoxieity from human studies are relevant to the mode of action for BZ-induced leukemogenesis, inhibjtion of topoisomerase II is the most likely mechanism. 3 BP-00011579 Benzene-Induced Genotoxlclty: A Different Perspective David A. Eastmond, Environmental Toxicology Program, University of California, Riverside, CA. 92521, USA -Although benzene is recognized as a human carcinogen, the critical mechanisms underlying its genotoxic and leukemogenic effects remain elusive. A large number of in vitro and in vivo genotoxicity studies of benzene and its metabolites have been conducted. However, given the requirement for metabolic activation, the complexity of benzene's metabolic pathways, and the potential for interactive effects, in vitro studies of benzene genotoxicity are currently of limited usefulness. Recently, a number of in vivo studies have been conducted using both conventional and recently developed molecular techniques which are providing valuable insights into the types of genetic alterations associated with exposure to high levels of benzene. The results of these animal and worker studies are largely consistent with earlier investigations employing more traditional approaches. These studies have shown that benzene is weakly effective in inducing specific gene mutations and binds inefficiently to bone marrow DNA. In contrast, similar studies have shown that benzene is effective in inducing large-scale chromosomal alterations. Chromosome breakage represents probably the-most common genetic lesion indtJced by benzene but other structural aberrations such as translocations can also be formed. Aneuploidy, a type of numerical chromosome aberration, is modestly increased with benzene exposure. This pattern of genotoxicity is consistent with that exhibited by other known human leukemia-inducing agents which have consistently been shown to induce chromosomal alterations as well as bone marrow toxicity. In addition. a recent update on two cohr::>rts.being followed by the European Study Group on Cytogenetic Biomarkers and Health (Hagmar et al., Cancer Res. 58:4117-21, 1998) has shown that individuals exhibiting elevated frequencies of chromosomal aberrations are at significantly elevated risk for developing cancer and that this risk increases with time. Similar types of clonal structural and numerical aberrations are seen in human leukemias and appear to play a critical role in leukemogenesis. This convergence of evidence indicates that-the induction of chromosomal alterations by benzene is likely to play a key role in its-genotoxic and leukemogenic effects. Although recent studieS-are making pr~s in identifying genotoxic mechanisms, much of this work is in its early stages and needs to be independently replicated. Identification of the metabolites contributing to benzene's genotoxic effects, the mechanisms underlying the alterations, and the specific chromosomes and chromosomal regions involved should be an important focus for future research. ----- - - - - - - - - - - - - - - - - BP-00011580 Benzene Leukemogenesis: An Environmental Carcinogen Induced Tissue Specific Model of Neoplasia using Genetically Altered Mouse Models: John Edgar French. Ph.D., NIEHS, NIH and Muriel Saulnier, DVM, NIEHS and NCSU-Raleigh, Research Triangle Park, NC 27709 USA Benzene is carcinogenic to laboratory rodents as well as to humans. However, the mechanism(s) ofbenzene toxicity and carcinogenicity are not well understood and there are no highly reproducible experimental rodent models for investigation that are not confounded by viral leukemogenesis. A major objective ofour laboratory is to develop carcinogen induced tissue specific models ofneoplasia. Critical toboth prospective identification ofcarcinogens and investigation ofcarcinogenic mechanism(s) is the induction of tumors within a sufficiently short time frame such that sporadic tumors will not confound investigation or interpretation due to aging-or attendant strain specific tumors. Ideally, tumor induction should be observed at tissue/organ sites consistent with transspecies carcinogens {i.e., concordant at common sites across species). In addition, a mode or a mechanistic basis, such as loss or mutations of a tumor suppressor gene or oncogene, for tumor induction should be easily and rapidly identified for use in risk assessment. Finally, to increase confidence in the extrapolation of data from rodent to human, the mode or mechanism must be consistent with our current knowledge ofhuman. Genetically altered mouse models, which have defined genetic lesions, which are critical but insufficient alone to induce cancer, are potential models for investigation ofchemical carcinogenesis and risk assessment. We are currently investigating benzene toxicity and carcinogenicity in different genetically altered mouse models: 1) FVB/N-Tg.AC (v-Ha-ras) mouse in which the transgene is inducible by both mutagenic and nonmutagenic carcinogens, 2) the C57BU6 or FVB/N-Trp53 mouse heterozygous for an inactivated p53 allele, and 3) the FVB/N-Trp53 heterozygous mouse hemizygous for the Tg.AC (v-Ha-ras) transgene. Benzene (>99.9% pure) was administered topically (0 or 200 J.d neat; 2x/da; 2x/wk for 14-26 weeks) or orally _(intubation) (0- 600 mglkg!BW; 5x/wk) for 26 weeks starting at 8 to 12 week-s of age to the selected lines plus coisogenic wildtype control mice. Oral administration ofbenzene to C57BL/6-Trp53 heterozygous mice resulted in the induction of sarcomas ofthe-subcutis (16/39), thymic 1ymphoma-(3/39), and a pancreatic carcinoma (l/30). No tumors were observed in the 20 vehicle treated controls. The frequent loss of the remaining wildtype p53 allele (LOH) was observed (13/16 sarcomas; 3/3 thymic lymphomas; 1/l pancreatic acinarcarcinoma). Oral administration ofbenzene to FVB/N-Tg.AC {v-Ha-ras) mice resulted in a dose related mortality and -bone marrow toxicity but no tumors. In three independent studies, topical application of benzene resulted in a dose-related induction of neoplasia (papillomas and spindle cell tumors) and granulocytic leukemia. Results from topical application ofbenzene to bigenic mice are currently under review. We conclude from these studies that there are differences in benzene toxicity and potential carcinogenicity depending upon the route ofadministration and the selection-ofthe test species strain. Furthermore,-benzene activation ofPKC and other signaling events lead to genetic events and resulted in skin and hematopoietic malignancies under these experimental conditions. These genetically altered mouse models may be suitable-for dose metric and mechanistic studies of benzene leukemogenesis by the inhalation route of exposure. BP-00011581 Myeloid Leukemia After Cytotoxic Therapy and Other Hematotoxinf.; Richard A. Larson, M.D Department of Medicine The Pritzker School ot Medicine and the Cancer Research Center, The-University of Chicago, Chicago, IL This work was supported by grants P01 CA-40046 and CA-14599 from the National Institutes of Health. Correspondence: Richard A. Larson, M.D The Univ-ersity of Chicago 5841 S. MarylandAve., MC 2115_ Chicago, IL 60637-1470 Phone: (773) 702-6783Fax: (773) 702-0963 E-mai-l: ralarson@mcis.bsd .uehicago.edu BP-00011582 Therapy-related leukemia is a-neoplastic hematopoietic disorder arising in most cases from a multipotent stem cell and in a few cases from a lineage-committed progenitor. Therapy-related acute myeloid leukemia (t-AML) describes a clinical syndrome that-exhibits important differences from AML that arises de novo-(-1). The terms "therapy-related" or "treatment-related" leukemia are descriptive and are based on a patient's history of exposure to cytotoxic agents. They imply a causal relationship, but the mechanism of this remains to be established. These terms may ultimately be too restrictive, since the leukemia that develops after exposure to benzene or atomic bomb irradiation is very similar or identical to the therapy-related leukemia syndrome. In the future, as various subtypes of leukemia are distinguished by specific genetic alterations, the terms de novo (or primary) and therapy-related leukemia will likely be discarded and specific etiologies incorporated into the diagnostic nomenclature. The development of therapy-related second neoplasms provides a unique, ethically acceptable environment for studying the effects of mutagens on carcinogenesis in man. The long latency period characteristic-of therapy-related cancers after initial mutagenic exposure suggests that "two hits" or perhaps multiple intermediate steps are required for full expression of the malignant phenotype. It has not been possible to determine whether the development of a tberapy-related neoplasm is a stochastic event, or whether certain individuals are at f:ligher risk (perhaps due to a DNA-repair deficiency or a heritable predisposition) and thus might be identifiable in advance (2). In geAeral, two paths of investigation have been expJored. The first involves meticulous clinico-pathological and cytogenetic analysis of individt1al cases as they present with therapy-related leukemia (3). The second involves large scale epidemiological surveys of patients at risk. There are now many such studies of each type in the literature. Patients with Hodgkin's disease were the first large cohort of relatively uniformly treated patients who experienced prolonged survival. After long-term follow-up. hundreds of cases of therapy-related leukemia have now been reported on and analyzed. It soon became clear that the risk of therapy-related leukemia was shared by 2 BP-00011583 patients with other cancers and even non-malignant disorders-if-they had received cytotoxic treatment (4). CLASSICAL THERAPY-RELATED LEUKEMIA In the classic form of therapy-related leukemia that follows treatment with alkylating agents and/or radiation therapy, the blood and bone marrow findings resemble those seen in the-primary myelodysplastic syndromes (MDS). Anemia and thrombocytopenia are extremely common. Leukopenia may also be present. Marked dysplastic changes are observed in all three cell lines. Early in the course of disease, red cell poikilocytosis may be particularly notable in the peripheral blood film. The bone marrow--may have variable cellularity, but is most often hypercellular. Hypocellular and even aplastic marrows are seen in some cases. Mild to marked reticulin fibrosis may be present. The degree of dysgranulopoiesis and dysmegakaryocytopoiesis is typically greater than that observed in primary-MOS. About two-thirds of patients present with fewer than 30% blast cells in the marrow and <5% blasts in the blood, and therefore, these patients have been diagnosed as tMDS. However, unlike primary MDS which may have a long preleukemic phase, patients with therapy-related leukemia have a more prominent arrest-of hematopoietic maturation and a more rapid accumulation of blasts. Typically, the t-MDS phase lasts for about sbc months. As these cases evolve to more overt leukemia, features characteristic of FAB subtypes M1, M2~ or M4 are most common. However, there are difficulties in classifyjng therapy-related leukemia according to the FAB criteria desined for AML de novo since most cases demonstrate trilineage involvement and often overlap several subtypes. Auer rods are rarely seen, and myeloperoxidase and non-specific esterase reactivity are often only weakly expressed. There is a continuum of clonate"Xpansion and dedifferentiation that occurs in the neoplastic clone and subclones. Shortened survival is more a function olfailur.e of normal hematopoiesis rather than _r:apid accumulation of bone marrow blast cells. 3 BP-00011584 Clonal chromosomal abnormalities, often-of a complex nature, are identified in mostcases of classical therapy-related leukemia (1-4). Loss of part or all of chromosomes 5 and/or 7 are the characteristic findings, and have been reported in overt 90% of cases in some series (3). Table 1 shows the distribution of cytogenetic abnormalities observed in 270 patients with therapy-related leukemia studied at the University of Chicago. The karyotypes are often complex. The most common single abnormality is monosomy 7, followed in frequency by deletion of the long arm of chromosome 5 [dei(Sq}]and by monosomy 5. These same abnormalities are observed in primary MDS and AML de novo, especially in older patients and those with occupational exposure to potential carcinogens. Their:. frequency, however, is clearly higher in therapy-related leukemia. Recent molecular investigation has focused on identifying a putative leukemia suppressor gene in chromosome band 5q31, a criticat-region that is consistently deleted in leukemia cells with 5q abnormalities. LEUKEMIA FOLLOWING TOPOISOMERASE II INHIBITORS Whereas classic t-AML is characterized by abnormalities involving the long arms of chromosomes 5 and/or 7, the leukemias secondary to agents that target topoisomerase II result in translocations involving the Mt.L gene on-chromosome 11, band q23, and less commonly, the AML1 gene on chromosome 21, band q22 (5}. At first.-the association was linked only to the epipodophyllotoxins, etoposide and teniposide. However, subsequent reports have also implicated DNA intercalating agents such as eloxorubicin. In contrast to classic t-AML, these leukemias have a much shorter latency between initiation of chemotherapy for the primary cancer and the development of leukemia (Table 2). In addition, a preceding myelodysplastic syndrome is not associated with these leukemias. The t(11q23) cases primarily have monoblastic (MS} or myelomonocytic (M4) pRenotypes, but cases of AML-M1 and M2 as- well as ALL have been described. The t(21 q22) cases are typically AML M2. 4 BP-00011585 DIFFERENT GENETIC MECHANISMS FOR LEUKEMOGENESIS The particular mechanisms of DNA damage that lead either to chromosomal deletions or to balanced translocations may underlie the differences in latencies between lbe two forms of therapy-related leukemia (5). In the case of chromosomal deletions, one allele of a putative tumor suppressor gene may be inactivated. Before the affected cell would gain a proliferative advantage, however, the second allele would also have to be deleted or mutated. Additionally, losses of both alleles of an individual tumor suppressor may not be sufficient to confer a malignant phenotype. As described in the model of color~ctal tumorigenesis, multiple tumor suppressor genes or oncogenes may need to be -mutated to ultimately transform -a-cell. This series of genetic changes may require an extended period of time, thus explaining the long latency of alkylator-induced t-AML. In contrast, balanced chromosome translocations result in the activatioA-of cellular oncogenes in a dominaot fashion. These rearrangements, such as those involving the MLL gene at 11 q23, may yield a fusion gene that acts as a dominant oncogene. Whereas this fusion gene alone may not be sufficient to transform an hematopoietic progenitor cell, relatively fewer genetic events may be required to proceed to the leukemic phenotype. In line with this hypothesis, 70-80% of all acute leukemias that occur de novo in infants, both lymphoid and myelotd; involve the MLL gene. Moreover, these cases have even been reported in the neonatal period. Thus, 11 q23 translocations can clearly induce the formation of leukemia over a short interval of time. The striking incidence of MLL_gene rearrangements in infant leukemias suggests a potential genetic susceptibility to translocations at this locus. If this were the case, perhaps only certain patients with an as yet unknown DNA repair deficiency might be susceptible to the mutagenic effect of topoisomerase II mhibitors. As the numbers of cancer survivors increase after conventional cytotoxic treatment, the incidence of therapy-related leukemia will undoubtedly l'"ise. As further understanding about mechanisms of mutagenesis develops, it is likely that-certain individuals-who have increased susceptibility to the leukemogenic activity of particular agents--ean be identified. I 5 BP-00011586 Individuals demonstrating benzene-associated hematotoxicity should undergo careful evaluation by clinical, morphological, cytogenetic, and molecular techniques in order to discover the intermediate steps in the progression to leukemia. REFERENCES Michael J. Thirman and Richard A. Larson. "Therapy-related myeloid leukemia." In: Stephen D. Nimer and David W. Gold-e, eds. Hematologic Complications of Cancer. Hematology/ Oncology Clinics of North America 1996; 10: 293-320. 2. RA Larson, M Banerjee, Y Wang, J Wiemels, C Hartford, MM LeBeau, MT Smith. "An inactivating polymorphism in the NAD(P)H: Quinone oxidoreductase (NQ01) gene in patients with primary and therapy-related myeloid leukemia(t-AML)." Proc Am Soc Clin Oncol1997; 16: 530a (abstract #1911 ). 3. LeBeau MM. Albain KS, Larson RA, et al: Ciinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes- and acute nonlymphocytic leukemia: Further evidence for characteristic abnormalities of chromosomes no. 5 and 7. J Clin 0Acol4:325-345, 1986 4. Levine EG, Bloomfield CD: Leukemias and myelodysplastic syndromes secondary to ckug, radiation,-and environmental exposure. Semin-oncol1992; 19: 471. 5. Pedersen-Bjergaard J, Rowley JD: The balanced and the unbalanced chromosome aberrations _of acute myeloid leukemia may develop in different ways and may contribute differently to ma1ignant transformation. Blood 1994; 83(1 0):2780-2786 I 6 BP-00011587 Table 1. Cytogenetic Abnormalities in Therapy-Related Myeloid Leukemia (University of Chicago series) No. of patients No. with clonal abnormalities Abnormalities of chromosomes 5 and/or 7 270 252 (93%) 191 (71%) Chromosome 5 only Chromosome 7 only Chromosomes 5 and 7 - t(11q23) 54 (20%) 76 (28%) 61 (23%) t(3;21) or t(6~21 }-or t(21-q22) t(15-;_:! 7) +8 inv(16) -13 or del(13q) Other clonal abnormalities 9 (3%) 8 (3%) 4 (1%) 7 (3%) 4 (1%) 3 (1%) 26 (10%) Data from MM LeB.eau, 1998 7 BP-00011588 Table 4;. Contr~sting Features of Ther~~py-Related Leukemia Secondary to Either Alkylating Agents or Topoisomerase 11 Inhibitors '' ' Chromosome Preleukemia FAB Morphology Latency Responsr to Long- Chemotherap;j I Abnormality Ph~se Induction term Drups Chemotherapy Survival Alkylating Agents del(5q), -5 d~l(7q), -7 MDS Topoisomerase II inhibitor 11q23 trarislocatlons 21q22 translocations none Various agents t(15;17) none Not classifiable by current criteria 5-7 years poor Usually M4, M5; 6 months- good some M1, M2 5 years and ALL-L1 ' M3 2-3 years good poor I poor good melphalan, mechlorethamine, chlorambucil, cyclophsophamide, carmustine, lomustine semustine procarbazine, dacarbazine, mitolactol etoposide, teniposide, actinomycin D, doxorubicin, 4 eRi-doxorubicin mitoxantrone bimolane inv(16) none M4Eo <3 years good good ttl ""0 I 0 ..0 0............ (J1 00 \0 THE INITIAL TRANSFORMING EVENT IN MYELODYSPLASTIC SYNDROMES MAY BE VIRAL Azra Raza, M.D. Professor o{Medicioe Director PreLeukemia and Leukemia Program Rush Cancer Institute Rush-Presbyterian-St. ~Luke's Medical Center Chicago, IL (USA) BP-00011590 SUMMARY; The myelodysplastic syndromes (MDS) are indolent clonal disorders which predominate in the elderly and present with the clinical paradox of variable cytopenias despite generally cellular bone marrows (BM) (1, 2). A novel paradigm has recently been proposed to explain the variegated nature of the pathology encountered in MDS (3). An as yet poorly defmed event(s) confers a growth advantage on a pluripotential BM stem cell whose proliferation eventually overwhelms thth-narrow leading to monoclonal hemopoiesis. In a certain percentage ofMDS patients, dual acting cytokines from unexplained sources further confound this picture by providing a stimulus to the early precursors to divide and inducing apoptotic death oftheir maturing progeny. It is important to note that apoptosis is found in all stages ofthe cell cycle, particularly-in cells actively synthesizing DNA (3). This situation ofapparent "antonymy" where a cell is found to be simultaneously inS-phase and apoptotic appears unique to MDS and could not be documented in a variety ofother cancers-such as head and neck, breast, ovarian, brain, lymphomas and de novo acute leukemias (3). Anyhow, this excessive proliferation-and apoptosis results in the clinical syndrome of pancytopenia (excessive intramedullary apoptosis of maturing cells) in the setting of hypercellular marrows (rapid proliferation ofthe transformed progenitors). The cytokines responsible for the genesis ofapoptotic death ofhematopoietic cells include tumor necrosis factor alpha (TNF-a), transforming growt.h-factor beta (TGF-B) and interleukin-1 beta (ILl-B) (4, 5). In some MDS patients, the-growth advantage conferred upon the stem cell may be matched by a propensity to undergo premature apoptosis as the cells differentiate. In still other patients, the genesis of cytopenias may not be excessive intramedullary apoptosis at all, but may be excessive retention of hematopoietic cells in the BM due to dysregulation of adhesion molecules and/or their receptors. What can be said with certainty is that hematopoiesisin MDS is monoclonal where both lymphoid and myeloid cells appear to be the progeny of the~ same parent (6-7). There is however, no incontrovertible proof that the "initial transforming event" (or series of events) occurs in a pluripotential BM stem cell in MDS. Rather, the diseased nature oTthe parent stem cell is inferred from the abnormalities manifest in its daughters, the most prominent being dysplastic maturation and the presence ofmonoclonal hemopoiesis. In fact, the reason why MDS is considered as representing a malignant state is because of its clonal nature and presence of- karyotypic abnormalities whichfrequently undergo evolution as the disease- progresses. Monoclonality in MDS should not be confused with monoclonality encountered in other malignant states-such as acute myeloid leukem-ia (AML). In the case of AML, the leukemia cells are clearly descended from a single transformed parent, however, residual hematopoiesis is polyclonal (8). In MDS on the other hand, cells belonging t-o-all three lineages (erythroid, myeloid, megakaryocytic) as well as probably the B- -2- BP-00011591 lymphocytes are monoclonal or descended from the single transformed parent cell (6). MDS resembles the chronic phase ofchronic myeloid leukemia (CML) where the normally differentiated (as opposed to dysplastic mature cells in MDS) erythroid, myeloid and megakaryocytic cells all contain the Philadelphia (Ph1) chromosome. The transforming event in CML appears to be the bcrabl genetic abnormality resulting from the Ph1 translocation since the BM returns to a polyclonal state upon regression ofthis cytogenetic anomaly following interferon therapy (9). 1n MDS however, even the frequently encountered cytogenetic abnormalities such as those affecting chromosomes 5 and 7 may be epiphenomena or secondary events since they often appear to be present in only a subpopulation of cells in a marrow which is otherwise monoclonal. In summary therefore, the earliest consequence of the initial transforming event in MDS is monoclonal hemopoiesis while cytogenetic abnormalities represent disease evolution. In CML on the other hand, absence of monoclonal-hemopoiesis upon eradication of the Ph1 chromosome appears to implicate the bcr-abl abnormality as the "transforming event". What Is The "Initial Event" In MDS? Since-there are certain karyotypic anomalies which occur with a regular frequency in MDS, it is not unreasonable to look for the "initial transforming event" in genic changes such as loss of a tumor suppressor gene or constitutive activity of a proto-oncogene associated with the most commonly involved areas of the affected chromosomes. However, even if such a gene was identified, the question ofwhat caused its loss or dysfunction would still remain obscure. In this paper we would like to propose that MDS can begin as a viral disease. We further propose that the virus may infect a BM stem cell, a BM stromal cell or a cell belonging to the immune system. Abnormal cytokine expression as a-consequence of the infection could change the bone marrow microenvironment in such a way that only a rapidly proliferating stem cell would survive in this new setting, thus accounting for the monoclonal- hemopoiesis seen in MDS. The daughters of such a rapidly proliferating abnormal stem cell might also undergoes premature apoptosis either as a result of the adverse microenvironment or as a direct consequence Dfincreased divisions in the parentsince it has been demonstrated that less than 10% of CD34+ cells dividing once in vitro were apoptotic as compared to 25% of CD34+ cells which had divided 4 or more times (1 0). Taken together, these abnormalities would produce the syndrome of monoclonal hemopoiesis, pancytopenia and hypercellular marrows. 1If this hypothesis is correct then the first essential question pertains to the identity of the virus. We propose that the virus is probably a conunon human pathogen that has developed long-term residence in the host without causing clinically significant ongoing disease. The virus however-is capable of reactivation from its l.1tency phase and -even be made "oneegenic" in the presence of the right -3- BP-00011592 "promoting conditions". These promoting events would include immunosuppression as a consequence of aging, bone marrow transplant or other chemo-toxic exposures, a second viral infection (acquired immune deficiency syndrome or AIDS) or fmally any other infectious and/or non-infectious conditions which lead to upregulation of pro-inflammatory cytokines. It is also worthwhile to remember that prolonged, chronic exposure to the promoting events probably carries far more oncogenic potential than short-lived surges of cytokines or exposure to cytotoxic agents. On the whole, it is important to emphasize tllat in the majority of patients with MDS, along with abnormalities of the BM stem cell, there also appears to be a dysregulation of the cytokine milieu in an otherwise exquisitely balanced and finely-tuned BM microenvironment In fact, if we suppose that the "initial event" in MDS is related to the BM microenvironment and dysregulated cytokines, then it is highly probable that the altered landscape would profoundly affect the fitness of the organisms that normally reside therein. For example, mutated progenitor cells such as those bearing the bcr-abl translocation or t(l4, 18) abnormalities have been clearly demonstrated in healthy individuals (11, 12), but probably remain at a low frequency because "normal" BM microenvironmental conditions are not conducive towards their expansion. In the changed environment however, these mutated or otherwise abnormal clones can conceivably acquire a growth advantage over their normal counterparts. Tills would eventually lead to monoclonalliemopoiesis, with a rapidly proliferating transformed parent whose daughters die prematurely either due to the action of excessive pro-inflammatory cytokines or as a consequence of increased divisions, or both. The syndrome of pancytopenia, hypercellular marrow, monoclonal hemopoiesis with excessive proliferation/apoptosis and-abnormal cytokines expression would ensue. This situation could persist as such with increasing deterioration of the existing clinical problems such as worsening cytopenias. On the other hand, acquisition of a mutation in one ofthe daughters ofthe transformed parent could lead to-loss of its ability to apoptose and mature, thereby giving rise to a new population of blastic cells and the transformation of a simple refractory anemia (RA) to refractory anemia with excess blasts (RAEB). Progression to acute myeloid leukemia (AML)-would depend on the rapidity wit.~ which these blast cells would be able to undergo substantial clonal expansion. Monoclonal hemopeiesis would clearly predispose to such accumulation of mutations in a daughter cell. During all ofthis disease causation and evolution, maintenance ofthe abnormal cytokines milieu in the BM microenvironment would be essential for disease persistence. A second possibility is obviously related to transformation ofa hematopoietic stem cell which would rapidly divide/apoptose and give rise-to abnormal cytokine production eventually leading-to the same scenario described above. A virus could accomplish-either ofthese possibilities by infecting an early BM progenitor cell or a BM stromal cell and we proposccthat a DNA virus belonging to the herpes virus family could be one such pathogen. -4- BP-00011593 REFERENCES; 1) Resogetti L. The nature and natural history of myelodysplasia. Haematologica 25(3): 191204, 199 2) Yoshida Y, Stephenson-], Mufti GJ. Myelodysplastic syndromes: From morphology to molecular biology. Part I. Classification, natural history and cell biology of myelodysplasia Int J Hematol 57:87-97, 1993. 3) Raza A, Mundie S, Shetty V, et al. A paradigm shift in myelodysplastic syndromes. Leukemia 10: 1648-1652, 1996. 4) Shetty V, Mundle S, Alvi-S, et al. Measurement ofapoptosis, fli'Oliferation and three cytokine in 46 patients with myelodysplastic syndromes. Leuk Res 20(211/12):891-900, 1996. 5) Mundie SD, Venugopal P, Pandav D, Broady-Robinson L, Gezer S, Robin EL, Rifkin SR, Klein M, Alston DE, Hernandez BM, Rosi D, Alvi S, Shetty VT, Gregory SA, Raza A: Indication of an involvement of interleukin=Ij3 converting enzyme (ICE)-like protease in intramedullary apoptotic cell death in the bone marrows of patients with myelodysplastic syndromes (MDS). Blood 88(7):2640-2647, 1996. 6) van Kamp H, Fibbe WE, -Jansen RPM, van der Keur M, de Graaff E, Willemze R, Landergent JE. Clonal involvement of granulocytes and monocytes, but not ofT and B lymphocytes and natural killer cells in patients with myelodysplasia: Analysis by X-linked restriction fragment length polymorphism and polymerase chain reaction of the phosphoglycerate kinase gene. Blood 80(7):1774-1780, 1992. 7) Janssen JWG, Buschle M, Layton M, Drexler HG, Lyons J, van den Berghe H, Heimpel H, Kubanek B, Kleihauer E, Mufti GJ, Bartram CR Clonal analysis of myelodysplastic syndromes: Evidence ofmultipotent stem cell origin. Blood 73(1):248-254, 1989. 8) Fialkow PJ, Singer PW, Adamson JW et al. Acute non-lymphocytic leukemia: expression in cells restricted to granulocytic and monocytic differentiation. N Engl J med 301:1-5, 1979. 9) Claxton D, Deisseroth A.-Talpaz M, Reading c. Kantarjian H. Trujillo J, Stass S, Gooch G, Spitzer G. Polyc1onal hematopoiesis in interferon-induced cytogenetic remissions ofchronic myelogenous leukemia Blood 79:997-1002, 1996. 10) Traycoff CM, Orazi A,-Ladd AC, RiceS, Mcmahel J, Srour EF. Proliferation-induced decline of primitive hematopoietic progenitor-cell activity is coupled with an increase inapoptosis of ex vivo expanded CD34+ cells. Exp-Hematol 26:53-62, 1998. -5- BP-00011594 11) Biemaux C, Loos M, Sels A, Huez G, Stryckmans P: Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 86(8):31183122, 1995. 12) Fuscoe JC, Setzer RW, Collard DD, Moore MM-:-Quantification of t(l4;18) in the lymphocytres of healthy adult humans as a possible biomarker of environmental exposure to carcinogens. Carcinogenesis 17(5):101-3-1020, 1996. I -6- BP-00011595 ROLE OF NITRIC OXIDE IN BENZENE INDUCED HEMATOSUPPRESSION Debra L. Laskin Environmental and Occupational Health Sciences Institute Rutgers University and UMDNJ/Robert Wood Johnson Medical School Piscataway, NJ It is becoming increasingly apparent that nitric oxide plays a multifunctional role in regulating inflammatory processes in the body. Although nitric oxide and its oxidation products are cytotoxic toward certain pathogens, they can also cause tissue injury, suppress proliferation and induce apoptosis. Cytokines and growth factors released at sites-of inflammation or injury stimulate both immune and nonimmune cells to produce nitric oxide. No where in the body is this more detrimental than in the bone marrow since the continuous production ofhematopoietic precursors is essential for normal blood cell maturation. Our laboratories have discovered that, in response to inflammatory mediators such as bacterial derived lipopolysaccharide (LPS), interferon-y (IFN y), rutd tumor necrosis factor-a (TNF a), bone marrow leukocytes, consisting predominantly of macrophages and-granulocytes readily produce-nitric oKide. This response is dependent on 1-arginine and blocked by the nitric oxide synthase inhibitors, monomethyl-1-arginine (L-NMA) and aminoguanidine. Western and northern blot analysis have revealed that bone marrow cell nitric-oxide pro_duction is mediated by the actions of an inducible form _ ofthe enzyme, nitric oxide synthase (NOS2). Additiona1 studies have demonstrated that nitric oxide production by bone marrow leukOGytes is augmented by hematopoietic growth factors including interleukin-3, macrophage colony stimulating factor, and granulocytemacrophage colony stimulating factor. Production of nitric oxide by bone marrow leukocytes is associated with impaired hematopoiesis, a response that is potentiated by colony-stimulating factors. T-!:eatment of mice with benzene, which-suppresses bone marrow cell development, markedly enhances the ability ofbone marrow cells to produce nitric oxide in response to inflammatory mediators alone and in combination with hematopoietic growth factors. Moreover, mice deficient in NOS2 as well as TNFa are protected form benzene induced bone marrow depression. Taken together, these data suggest that nitric oxide may be an important mediators ofbenzene-induced bone marrow suppression (Supported by NIH grants ES06897 and ES0502-2 and the Burroughs Wellcome Fund). References: MacEachern, L. and Laskin. D.L. (1992). Increased production of tumor necrosis factor by bone marrow leukocytes following benzene treatment of mice. Toxicol. Appl. PbarmacoL, 113: 260-266. BP-00011596 Punjabi, C., Laskin. D.L., Heck, D. and Laskin, J.D. {1992). Production ofnitric oxide by bone marrow cells: Inverse correlation with cellular proliferation. J. Immunol., 149: 21792184 MacEachern, L., Snyder, R. and Laskin. D.L. (1992). Alterations in the morphology and functional activity ofbone marrow phagocytes following benzene treatment ofmice. Toxicol. Appl. Phannacol., 117: 147-154. Punjabi, C., Laskin, J.D., Hwang, S.M., MacEachern, L., and Laskin, D.L. (1994). Enhanced production ofreactive nitrogen intermediates by mouse bone marrow cells and increased sensitivity to M-CSF is associated with induction of nitric oxide synthase gene expression. Blood, 83: 3255-3263. Laskin, J.D., Rao, N.R., Punjabi, C.J., Laskin, D.L. and Snyder, R. (1-995). Distinct actions of benzene and its metabolites on nitric oxide production by bone marrow leukocytes. J. Leukoc. Bioi., 57:422-426. Laskin, D.L., Heck, D.E., Punjabi, C.J., and Laskin, J.D. {1996). Role ofnitric--axide in hematosuppre;ssionand benzene induced toxicity. Env. Health Persp. 104:1283-1287. BP-00011597 Molecular Models of Benzene Leukemogenesis. Richard D. Irons, Molecular Toxicology and Environmental Health Sciences Program, Dept of Pharmaceutical Sciences, School of Pharmacy; Dept. ofPathology, School ofMedicine; and Comprehensive Cancer Center, University ofColorado Health Sciences-Ctr., Denver, CO., USA. Several years ago, we proposed a multistep model for the evolution of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) developing secondary to drug or chemical exposure. The model described a multistep progression in which a relatively small number ofalternative pathways converge in the development ofleukemia. Its essential elemeats were as follows: Chemical-induced transient changes in cytokine response result in altered regulation ofproliferation in the hematopoietic target cell compartment; Increased proliferation predisposes to development ofan early clonal chromosomal abnormality, primarily involving deletion ofall or significant-regions of individual chromosomes, most often involving chromosomes 5 and 7; Interaction of the emerging-clone with the hematopoietic stromal environment leads to clonal selection and outgrowth; Additional random mutational events occurring in dividing cells predispose to further selection resulting in the ultimate development of the leukemic phenotype. We have found the model to be a useful tool for use--in integrating hematopoietic paradigms for the origin and progression of MDS/AML with expanding information on the patterns and molecular-events associated with secondary AML. Since its inception, descriptive, mechanistic and clonal data has emerged to both reinforce and amplify various aspects of the model architecture, as well as to pose additional questions with respect to-the specific applicability of the model to benzene leukemogenesis. This presentation will outline and review the model in light of the current --state of knowledge with respect to its applicability to benzene. Mechnanistic and cytogenetic findings from our laboratory and others will be discussed BP-00011598 with respect to their impact on the model and in assessing its relevance to benzene. Finally, these results will be evaluated with a view toward identifYing the types of information that would be most useful in further refinement and validation ofa benzene leukemia model as well as in critically evaluating endpoints ofinterest as potential biomarkers ofbenzene exposure. BP-00011599 1 Utility of a Mouse Model for Studying the Effects of Benzene on the Myeloid -Lineage. George F. Kalf, Ph.D. Department of Biochemistry and Molecular Pharmacology and the Kimmel Cancer Institute, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA. It was previously demonstrated in this laboratory (1) that the acute administration to mice of benzene or hydroquinone (HQ), a metabolite found in the bone marrow, causes a severe depression in the number of cells of the bone marrow except those of the granulocytic lineage which not only survive, but differentiate and increase in number. Renz and Kalf (2) provided an explanation for this observation by the demonstration that in mice benzene in vivo or HQ in vitro caused an inhibition of the production of interleukin-1 by stromal macrophages. lnterleukin-1 is obligatory for the induction of cytokine production by stromal fibroblasts without which the progenitor cells in the bone marrow do not survive. These results-suggested that the granulocytic progenitor cells survive and differentiate because HQ can substitute for granulocyte-colony stimulatiAg factor (G-CSF), the requisite cytokine for granulopoiesis, and induce differentiation in the myeloid stem cell. The ability to alter cytokine-dependent growth and differentiation jn hematopoietic- progenitor cells appears to be a property of agents that have a potential for causing secondary leukemia in humans. It was important to know- whether HQ had a deleterious effect on cytokine-induced proliferation and/or differentiation of the restricted myeloid stem cell, the myeloblast, and if so, whether HQ could effect any of the changes typically seen in the phenotype of leukemia cells in culture. We utilized the continuous IL-3-dependent mouse cell line, 320 clone 3(G) as our model system (1,4). This cell line was derived by Greenberger et al. (3) from normal bone marrow of C3H/HeJ mice. It has many features that allowed us to detrumine whether HQ was affecting myeloid differentiation by interacting with essential cytokines. The cell line is: 1) myeloblastic and-has a normal karyotype; 2) nonleukemic as indicated by its inability to cause tumors in nude mice or to generate cytokine-independent clones; 3) IL-3-dependent for survival and prollieration; and 4) is induced to undergo terminal granulocytic differentiation in the-presence of the physiological inducer of the granulocytic lineage, G-CSF. Myefub!asts were cultured in the presence of a concentration of IL-3 low enough to sustain survival and a level of proliferation that would not overly compete- with the induction of differentiation by G-GSF or HO- (1 ,4y. Morphological assessment of granulocytic differentiation was performed on Cytospin-preparecl, May-Grunwald/Giemsa-stained cell monolayers plated on polylysine-coated ~ass slides. Differentiation. was expressed as the percentage of total cells showing some stage-of granulocytic mor-phology. Total differentiat~an was defined as the combined percentages ef- promyelocytes, myelocytes, metamyelocytes and banded/segmented, or terminally differentiated ceHs, and BP-00011600 2 was detennined by averaging the percentages of total differentiation out of 200 cells on each of triplicate slides. Terminal differentiation was assessed by counting only segmented granulocytes. The induction of terminal granulocytic differentiation was verified by demonstrating that G-CSF or HQ caused the induction of various functional properties of granulocytes such -as superoxoxide production (measured as nitroblue tetrazolium (NBT) reduction), the expression of specific enzymes (i.e. chloroacetate esterase), and the appearance of granulocytic-specific cell s~;~rface antigens. No monocytes were induced. IL-3, while sustaining survival and proliferation of the myeloblasts, did not induce differentiation. HQ {1-2 !JM} was shown to induce total granulocytic differentiation in comparison with G-CSF as assessed by morphology and NBT reduction (1 ,4}. The putative inducing agent appears to be benzoquinone (BQ) from the myeloperoxidase-mediated oxidation of HQ in the myeloblast since: 1) resorcinol, a non oxidizable structural analog of BQ that does not serve as a substrate for myeloperoxide, does not induce differentiation, and; 2) the concomitant presence of indomethacin, a peroxidase inhibitor, with HQ inhibits HQ-induced differentiation. G-CSF effects the initiation of differentiation in the myeloblast by the upregulation of the 5-lipoxygenase (LPO) pathway for the production of leukotriene 0 4. an essential intracellular mediator of G-CSF transmembrane signaling. With the use of highly specific LPO inhibitors it was determined that BQ did not affect the LPO pathway for the production of LTD4 in the myeloblast {4). Because of the ability of BQ to interact covalently with proteins containing sulfhydryl groups, it was anticipated that BQ might induce granulocytic differentiation in myeloblasts by activating the LTD4 receptor thus obviating the requirement for LTD4. Concomitant addition of the specific receptor antagonist, MK-571, with HQ showed a concentration-dependent inhibitien of iRduction of differentiation by both HQ and G-CSF. LTD4 was shown to replace G-CSF in a concentration-dependent (1 0'9 to o1 5 M) induction of granulocytic differentiation. HQ showed a qualitatively similar concentration-dependent 5X1 0'7 to 4X1 o-s M) induction of differentiation allowing the comparison of the nature of the differentiation induced by the binding of the two ligands to the receptor. Competition studies involving the addition of various concentrations of receptor antaonist either prior to or after treatment of the cells with HQ or LTD4 suggest toot BQ binds more tightly to the receptor than the specifjc antagonist. G-CSF, LTD4 and HQ each induced about 97% total differentiation. Terminal differentiation as a percentage of the total is about 50% in the case of G-CSF and LT04, whereas-HQ only induced about 15 % tenninal differentiation. The fact that HQ and LTD4 both putatively work through the same receptor, but cause significantly different levels of terminal differentiation led us to focus on a comparison of the consequence of the interaction of the two ligands with the receptor. We carried out a morphological analysis of the kinetics of stage-specific granulocytic-differentiation over a 6-day period induced by HQ in comparison with LTD4. LTD4-induced differentiation showed a predominance of mature, BP-00011601 3 segmented granulocytes at Days 2 through 6, whereas, HQ-treated myeloblasts showed an incomplete differentiation profile at each day studied that was arrested at the myelocyte stage and showed very few mature granulocytes. o-sReceptor antagonist {-1 M} was capable of competing effectively with G-CSF or LTD4 for the receptor as measured by total differentiation induced, however, it could not compete with HQ suggesting that BQ may be binding c.ova:Jently to the receptor and thus constitutively activating it. HQ in the presence of G..csF or LTD4 caused a shift in the differentiation-profile from one of terminal granulocytes to one of immature myelocytes. This result suggests that in the bone marrow, where HQ and G-CSF may be present at the same time, the pattern of terminal granulocytic differentiation will be shifted to that of predominately an incomplete differentiation to myelocytes that retain proliferative capacity. If HQ, -at concentrations that induce granulocytic differentiation, is added to a culture of myeloblasts growing in the presence of IL-3, a 2 to 3-fold increase in the number of myeloblasts is seen (5}. We have determined that this increase does not result from an ability of HQ to deliver a proliferative signal to the myeloblast or from its ability to synergize with IL-3 to increase its proRferative signal. In the-absence of IL-3, HQ did not stimulate cell growth, but did-delay the death of IL-3-deprived myeloblasts. If IL-3 is withdrawn from the myeloblast, -so% of the cell population undergoes programmed cell death within 48 hours as determined by the observation of morphological changes such as cytoplasmic condensation, cell volume r-eduction, vacuolization, densely staining micronuclei, apoptotic bodies, and the presence of intemucleosomal DNA fragments observed as a DNA ladder in a gel. The relevant question became whether the increase in the number of myelocytes that occurs when HQ ~induces differentiation can be attributed to the proliferative capacity of the myelocyte or to an ability of HQ to prevent or delay apoptosis. HQ at concentrations (1-2J.!M) that induce differentiation in myeloblasts were shown to prevent apoptosis induced by either IL-3 withdrawal or treatment with staurosporine-(5) We have previously shown (6) that BQ _inhibits the activity of IL-1 converting enzyme (1CE), a member of a family of caspace proteases involved in the final- steps in the regulation of apoptosis. In the myeklid lineage, the caspace involved is caspase 3. BQ (2J.!M) was shown to inhibit recombinant caspace 3 by 50 per cent in a kinetic assay involving a specific fluorescent peptide containing the specific active site sequence of the caspace family of proteases. These results suggest that HQ via conversion to BQ prevents apoptosis in a proliferating population of early myeloidprogenitors. In summary: 1) Benzene and HQ induce an altered pattern of granulopaiesis in the mouse; 2) In a model culture system, HQ by binding to and constitutively activating the normal signal-driven LTD4 receptor, induces an incomplete differentiation pattem in myetoblasts where the progenitor cell is the proliferating myelocyte; 3) HQ can compete with the physiological inducer, G-CSF, in the induction of terminal granulocytic differentiation in the- myeloblast such that the differentiation profile is changed from one of mature segmented granulocytes to that of immature myelocytes; 4) HQ, by inactivation of an BP-00011602 4 essential caspace can prevent programmed death in myelocytes. Taken together, these results indicate that, in the presence of HQ in culture or in the bone marrow, myeloblasts can be induced to differentiate into an expanding clone of early progenitors that Jack the capacity for te-rminal differentiation and thus have two phenotypic pmperties of leukemic cells in culture. The use of mouse myeloblasts as a model system provides an ability to study epigenetic changes-resulting from exposure of myeloblasts- to various metabolites of benzene and-to compare the results with those obtained in tMa ben~ne-treated mouse mouse or alternatively to verify those obtained with myeloblasts by testing in the mouse. 1. Benzene aRd its metabolite, hydroquinone, induce granulocytic differentiation in -myeloblasts by interacting with cellular signaling pathways activated by granuJocyte colony,..stimulating factor. (1995), Hazel, Betsy, O'Connor, Annette, Nicu!escu, -Rodica and Kalf, George. Stem Cells, 13: 295-310. 2. Role for interleukin-1 (IL-1) in benzene-induced hematotoxicity: Inhibition of conversion of pre-IL-1alpha to mature cytokine in murine macrophages by hydroquinone and prevention of benzene-induced hematotoxicity in mice by IL-1alpha. (1991) Renz, John and KaU, George, Blood, 78:938-944. 3. Demonstration of permanent factor-dependant multipotential hematopoietic cell lines. (1983), Greenberger, J., Sakakeeny, M. Humphries, R., et al., Proc. Nat[.Acad. Sci. USA,-80:2931-2935. 4. Induction of granulocytic differentiation in myeloblasts by hydroquinone, a metabolite of benzene, involves the leukotriene D4 receptor. (1996), Hazel, Betsy and Kalf, George. Receptors and Signal Transduction, 6:1-12. 5. Inhibition of the conversion of pre-interleukins-1-alpha and 1beta to mature cytokines by p-benzoquinone, a metabolite of benzene. (1995), Niculescu, Rodica, -B-radferd, Harlan, Colman, Robert, and Kalf, George. Chem-Biol. Interact. 98:211-222. 6. Hydroquinone, a metabolite of benzene inhibits apoptosis in myeloblasts. (1996) Hazel, Betsy, Baum, Christine, and Kalf, George. Stem cells. 14:730- 742. BP-00011603 Benzene and Lymphohematopoietic Malignancies in China. Richard B. Hayes, PhD Cancer risk was assessed in a large cohort ofbenzene-exposed workers in China(l-3). Translational studies were also carried out among workers with current exposure to benzene (4) and among workers with a history of benzene poisoning (5), to provide insight into benzene carcinogenesis in humans. Cohort study ofbenzene-exposed workers. The cohort of benzene exposed workers was comprised of men and women employed between 1972-87 in 1,427 selected-benzeneexposed work units (departments) in 672 factories in 12 cities in China. A variety of industries and occupations using benzene were studied, including painting, printing, and manufacture_offootwear, paint, and other chemicals. An unexposed comparison group was assembled from workers-employed between 1972-87 in work units where benzene was not used in 69 of these factories and in 40 additional factories.(6,7) Subjects were identified from salary records and other factory written administrative records. We abstracted demographic data,including name, birth date, sex, and occupational data, including the dates of employment, by work unit and job title, for all jobs held by subjects in the study factories. FaF benzene exposure assignment, we developed a standardized job-title dictionary comprised of 60 benzene exposure-specific job-titles in 11 major activity groups. Exposure assignments were made for benzene-exposed jobs by a factory exposure assessment team consisting ofindustrial hygienists, factory safety officers, and other -employees, following a predetermined exposure assignment algorithm. Using available air measurements of benzene (n = 8,477) and abstracted data on benzene use and working conditions, benzene exposure was assigned in six-concentration ranges (<1 ppm; 1-5 ppm; 6-10 ppm; 11-25 ppm; 26-50 ppm; and >50 ppm) for each job in seven calendar periods (1949-59, 1960-64, 1965-69, 1970-74, 1975-79, 1980-84, 1985+). Following exposure assignment, data abstraction forms were edited and reviewed to resolve discrepancies between benzene-exposure estimates-and abstracted exposure informati9n. Subjects were followed up for history ofbenzene toxicity, selected lymphohematopoietic malignancies and other hematologic disorders and for vital status to December 31, 1987 through factory personnel records at study factories and subsequent places of employment, or, as needed, by contacting next-of-kin, work colleagues, treating physicians, or others. For deceased subjects, cause ofdeath was obtained from employer medical records, other written factory records or death certificates. Only after extensive search had failed to locate written records listing cause ofdeath, were treating physicians or next-of-kin contacted. For cases newly diagnosed with lymphohematopoietic malignancies and other hematologic disorders during the period of follow-up, pertinent histopathologic material, pathology reports and medical records were requested. For pathology-review, clinical, laboratory, and pathologic data were abstracted onto standardized fonns by physician investigators~- BP-00011604 All histopathologic and bone marrow aspirate slides and peripheral blood smears were reviewed systematically, by expert hematopathologists affiliated with the Mayo Clinic and Peking Union Hospital, using structured abstract forms to objectively characterize hematopoiesis. Diagnoses were assigned after evaluation ofall available clinical, laboratory, and pathologic data, without knowledge ofthe patient's benzene exposure status. Published criteria were used to categorize leukemia and lymphoma cases, and where possible to classify these cases and those with myelodysplasia (MDS).(8,9) For selected analyses, AML and MDS were combined as one disease category because of possible commonalities in the natural history ofthese conditions, and-failure in the past to consistently classify MDS as entities distinct from AML. For the statistical analysis, person-years were accumulated for the benzene-exposed workers from January 1,1972 or, ifhired later, from the first date of employment in a benzene-exposed job. For the unexposed-comparison group, person-years were accumulated from January 1, 1972 or, if hired later, from the first date of employment. Analyses for mortality due to all causes and for incidence oflymphohematopoietic malignancies and other hematologic disorders were made by internal comparison of disease rates in the benzene-exposed group to the rates in-the unexposed group, by Poisson regression analysis, yielding rate ratios (RR) for exposed versus unexposed workers, with control for age and, where appropriate, for sex. Dose-response was assessed with respect to duration (<5, 5-9, 10 +years), average (<10, 10-24, 25 +ppm), and cumulative exposure (<40, 40-100, 100 + ppm-yrs) to benzene. For dose-response analyses, each measure of benzene exposure for each individual was allowed to change with time; person-years and disease events were assigned to benzene exposure levels with a 1.5 year lag (i.e., according to the level 1.5 year previously). In order to further evaluate the temporal component of disease development. we partitioned cumulative exposure at a given time into recent (1.5 years to 10 years earlier) and distant (1 0 or more years earlier) exposure. Because a substantial number of workers were exposed to a relatively stable average amount ofbenzene, we also developed a measure of constant exposure, where follow-up was censored 1.5 years after the individual's exposure level changed {< 10 ppm, 10-24 ppm, 25 + ppm) for the first time. The stu!y group consisted of74,818 benzene-exposed and 35,805 unexposed workers. On average, benzene-exposed subjects were followed-for 10.5 years, while unexposed subjects were followed for 11.7 years. Women contributed 47 percent ofthe person-years in the benzene--exposed study greup and 40 percent in the unexposed group. Overall, the study groups were young, with about 60 percent ofthe total person-years at risk being contributed by subjects less than thirty years of age at stugy entry. About 2 percent of study subjects died during the follow-up period (1,369 benzene-exposed and 598 unexposed). Only 147 exposed and 90 unexposed workers were lost to follow-up. There were 412 benzene poisoning cases among 62,234 exp9sed subjeets (614,509 person-years) in 11 of the study cities. Risks for benzene poisoning increased with increasing intensity ofbenzene exposure at one and a half year prior to diagnosis of BP-00011605 benzene poisoning(< 5 ppm, RR = 1 [referent]; 5-19 ppm, RR = 2.2 (95%CI=l.7-2.9); 20-39 ppm, RR = 4.7 (95%CI=3.4-6.5); and 40 +ppm, RR = 7.2 (95%CI=5.3-9.8). Relative risks ofbenzene poisoning by cumulative exposure of 40-99 ppm-yrs, 100-399 ppm-yrs, and 400 and mor-e ppm-yrs, respectively, were 1.7 (95%CI=l.3-2.3), 2.0 {95%CI=1.5-2.6), and-2.4 (9So/oCI=1.8-3.2), compared to subjects who had less than 40 ppm-yrs cumulative exposure to benzene.{I0) Eighty-one incident cases oflymphohematopoietic malignancies {n= 63) and other hematologic disorders (n"" 18) were documented among benzene-exposed workers, with 13 lymphohematopoietic malignancies and no other hematologic disorders identified among the unexposed comparison group (1). Risk was significantly elevated for all lymphohematopoietic malignancies combined (RR= 2.6), malignant lymphoma (RR= 3.5) and leukemia (RR= 2.6). Among the leukemia subtypes, only AML was significantly elevated (RR= 3.1), although non-significant excesses were also noted-for CML (RR= 2.6) and lymphocytic leukemia (RR= 2.8), the latter attributable to 5 eases of acute lymphocytic leukemia. Significant excess risks were also found for aplastic anemia and myelodysplastic syndrome. The relative risk for the rubric ofacute myeloid leukemia plus MDS was RR= 4.1. The relative risk for alllymphohematopoietic malignancies and other hematologic disorders combined was 3.4 and for non=Hodgkin!s-lymphoma the RR= 3.0. No cases of Hodgkin's disease were identified. Disea-se risks were similar for men and women (11). Risk for ANLLI MDS was substantially increased {RR= 70.6), however, among workers with a prior history of.benzene poisoning (51. Although occupation-specific results were limited by small numbers, risks for ANLLIMDS were elevated among coatings workers (RR= 4.2), rubber workers (RR= 6.1), chemical workers (RR= 4.5), and among those with other or mixed-occupations (RR= 4.4) (3). Risk for NHL was also elevated among several occupational groups, but only chemical workers showed-a statistically si-gnificant excess (RR=~8). ANLL and ANLL/MDS both showed-patterns of increasing risk with increasing average exposure to benzene, with more consistent exposure-response patterns for ANLL/MDS than for ANLL alone (3). The link of ANLLIMDS with average exposure was strongest when restricted to subjects with constant levels of exposure: risks rose from 3.2 for those with constant low level exposures (< 10 ppm) to 7.1 for those with constant high level exposures (25 +ppm). Risk for ANLLIMDS increased with increasing.cumulative exposure to benzene, but the highest risks were not seen in the highest exposure level (cumulative ppm-years: 40-99, RR= 6.0; cumulative ppm-years: 100 +, RR= 4.4). Although there was some evidence of increased risk fOr leukemias other than ANLL among benzene-exposed workers, clear patterns of increasing risk with increasing exposure were not observed. Risk of ANLLIMDS was six-fold and significantly incr-eased among those who had only recent benzene exposure (14 exposed cases). Risk ofANLLIMDS was also strongly -I associated with-increasing amounts of recent (p for trend= 0.003) but not distant exposure (p for trend= 0.51 ). NHL was strongly linked with distant exposure to benzene (p for BP-00011606 trend= 0.005, 13 exposed cases), but the association with exposure in the most recent 10 years was weak (p for trend= 0.15, 16 exposed cases). Translational studies. Selectedoiamarkers were assessed among workers heavily exposed to benzene (n = 44; median benzene exposure: 31 ppm as an 8-hour TWA), among workers with a history of benzene poisoning (n= SO; benzene poisoning based upon-routine hematologic exams), and amongunex:posed controls, in Shanghai, China. Workers currently exposed to benzene had lower counts for white blood cells, lymphocytes, platelets, and red blood cells, and a lower hematocrit (4), however, lymphocyte count was the most sensitive indicator ofbenzene-induced hematotoxicity. Benzene air levels correlated with urinary levels ofphenol, muconic acid, hydroquinone, and catechol. however, among those with the highest exposure to benzene, hydroquinone and muconic acid levels tended to plateau as a proportion of benzene metabolites. Workers with a history ofbenzene poisoning were more likely than comparison controls to be rapid excretors ofchlorzoxazone, an indicator ofCYP2El activity, and to have two copies ofthe NQOI 609C-T mutation (combined OR= 7.6) (5). CYP2El and NQOl enzymes are involved. respectiv~ly, in activation and detoxification of benzene an<i-its metabolites, suggesting that genetic determinants ofbenzene metabolism may influence risk ofbenzene poisoning, which itselfis a risk factor in our cohort study for ANLLI MDS (RR= 70.6). Acknowledgments. My collaborators in this work are: Drs. S-N Yin, G-L Li and L-Q Xi ofthe Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, China, Drs. Y-Z Wang and W Lu of the Station of Public Health and Prevention oflnfection in Shanghai, China, Dr. Z-L Jiang of the Institute ofLabor Health and Occupational Disease in Tianjin, China, Dr.-T-R Dai of the Station of Public Health and Prevention of Infection in Chengdu, China, Dr. W-U Zhang of the Institute ofLabor Health and Occupational Disease in Sichuan, China, Dr. X-J Chao of the Station of Public Health and Prevention of Infection in Chonqing, China, Dr. P-Z Ye ofthe Institute of Labor Health and-Occupat~onal Disease in Heilongjiang, China, Dr. Q-R Kou of the Institute ofLabor Health and Occupational Disease in, Shenyan, China, Dr. Y-H Fan of the Institute ofLabor Health and Occupational Disease in Jinzhou, China, Dr. X-C Zhang ofthe Institute efLabor Health and Occupational Disease in Henan, China, Dr. X-F Lin of the Institute ofLabor Health and Occupational Disease in Guan-gzhou, China, Dr. J-F Men-g of the Institute ofLabor Health and Occupational Disease in Jiangx-i, China, Dr. J-S Zho of the Institute of Labor Health and Occupational Disease in""Nanchang, China, Dr. WJ Blot ofthe International Epidemiology Institute, Rockville, MD, Drs. MT Smith, L-P Zhang,-S Campleman and N Titenko-Holland of the University of California, Berkeley, CA. Drs. Z-N Zhang and D-G Li of the Peking Medical College Hospital, Beijing, China, Dr. C-Y Li of the Mayo Clinic, Rochester, Minn, Drs DRoss and RD Traver of the University of Colorado, Denver, CO,, Dr. B Hoener oftbe University ofCalifornia, San Francisco, CA, Dr. W Bechtold ot the Lovelace Inhalation Toxicology Research unit, Albuquerque, NM, and Drs. M Linet, N Rothman, S Wacholder, R N Hoover, W-H Chow BP-00011607 and LB Travis ofthe Division ofCancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD. BP-00011608 - - - - - - - - - - - - - - - - - - - - - - -- Bibliography l. Yin. S.N., Hayes, R.B., Linet, M.S., Li, GL., Dosemeci, M., Travis, L.B., Li, C.Y., Zhang, Z.N., Li, D.G., Chow, W.H., Wacholder, S., Wang, Y.Z., Jiang, Z.L., Dai, T.R., Zhang, W.Y., Chao, X.J., Ye, P.Z., Kou, Q.R, Zhang, X.C., Lin. X.F., Meng. J.F., Ding, C.Y., Zho, J.S., and Blot, W.J. A cohort study ofcancer amang benzene-exposed workers in China: overall results. Am J Ind Med, 29: 227-235, 1996. 2. Hayes, R.B., Yin. S.N., Dosemeci, M., Li, G.L., Wacholder, S., Chow, W.H., Rothman, N., Wang, Y.Z.,Dai, T.R., Chao, XJ., Jiang, Z.L., Ye, P.Z., Zhang, X.C., Kou, Q.R., Zhang, W.Y., Meng, J.F., Zho, J.S., Lin, X.F., Ding, C.Y., Li, C.Y., Zhang, Z.N., Li, D.G., Travis, L.B., Blot, W.J., andLinet, M. Mortality among benzene-exposed workers in China. Environ. Health Perspect. 104: 1349--1352, 1996. 3. Hayes, R.B., Yin, S.N., Dosemeci, M., Li, G.L., Wacholder, S., Travis, L.B., Li, C.Y., Rothman, N., Hoover, R.N., and Linet, M.S. Benzene and the dose-related incidence of hemato1ogic neoplasms in China. J. Natl. Cancer lost 89: 1065-1071, 1997. 4. Rothman, N., Li, G.L., Dosemeci, M., Bechtold, W., Marti, G.E., Wang, Y.Z., Linet, M., Xi, L.Q., Lu, W., Smith, M.T., Titenko-Holland, N., Zhang, L.P., Blot, W.J., Yin. S.N., and Hayes, R.B. Hematotoxicity among workers h-eavily exposed to benzene. Am J Ind Med, 29:236-246, 1996. 5. Rothman, N., Smith, M.T., Hayes, R.B., Traver, R.D., Hoener, B., Campleman, S., Li, G.-L., Dosemeci, M., Linet, M., Zhang, L., Xi, L., Wacholder, S., Lu, W., Meyer, K.B., Titenk:o-Holland, N., Stewart, J.T., Yin, S., and Ross, D. Benzene poisoning, a risk factor for hematological malignancy, is associated with NQOJ 609C-T-mutation and rapid fractional excretion ofchlorzoxazone. Cancer Res. 57: 2839-2842, 1997. 6. Yin, S.N., Linet, M.S., Hayes, R.B., Li, GL., Dosemeci, M., Wang, Y.Z., Chow, W.H., Jiang, Z~L., Wacholder, S., Zhang, W.U., Dai, T.R., Chao, X.J., Zhang, X.G., Ye, P.Z., Kou, Q.R., Meng, J.F., Zho, J.S., Lin, X.F., Ding, C.Y., Kneller, R., and Blot, W.J. Cohort study among workers exposed-to benzene in China. I. General methods and resources. Arn.J.Ind.Med. 26: 383-400, 1994. 7. Dosemeci, M., Li, G.-L., Hayes, R.B., Yin, S.-N., Linet,M-., Chow, W.-H., Wang, Y.-Z., Jiang, Z.-L., Dai, T.-R., Zhang, W.-U., Chao, X.-J., Ye, P.-Z., Kou, Q.-R., Fan, Y.-H.,Zhang, X.-C., L~ X.-F., Meng,.l..-F., Zho, J.-S., Wacholder._S., Knellez:, R., and Blot, W.J. A cohort study among workers exposed to benzene in China. II. Exposure assessment Amer J Ind Med, 26:401-411, 1994. 8. Travis, L.B., Li, C.Y., Zhi, Z.N., Li, D.G., Yin, S.N., Chow, W.H., Li, G.L., Dosemeci, M., Blot, W., Fraumeni, Jr., Hayes, R.B., and Linet, M.S;-Hematopoietic malignancies and related disorders among benzene-exposed workers in China. Leukem Lymph, 14: 91-102, 1994. BP-00011609 I 9. Linet, M.S., Yin, S.N., Travis, L.B., Li, C.Y., Zhang, Z.N., Li, D.G., Rothman, N., Li, G.L., Chow, W.H., Donaldson, J., Dosemeci, M., Wacholder, S., Blot, W.J., and Hayes, R.B. Clinical features of hematopoietic malignancies_and related disorders among benzene-exposed workers in China. Environ. Health Perspect.J04: 1353-1364, 1996. 10. Dosemeci, M., Yin, S.N., Linet, M., Wacholder, S., Rothman, N., Li, G.L., Chow, W.H., Wang, Y.Z., Jiang, Z.L., Dai, T.R., Zhang, W.U., Chao, X.J., Ye, P.Z., Kou, Q.R., Fan, Y.H., Zhang, X.C., Lin, X.F., Meng, J.F., Zho, J.S., Blot, W.J;, and Hayes, R.B. Indirect validation ofbenzene exposure assessment by association with benzene poisoning. Environ. Health Perspect.J04: 1343-1347, 1996. II. Li, G.L., Linet, M.S., Hayes, R.B., Yin, S.N., Dosemeci, M., Viang, Y.Z. Chow, W.H., Jiang, Z.L., Wacholder, S., Zhang, W.U., Dai, T.R., Chao, X.J., Zhang, X.C., e, P.Z., Kou, Q.R., Meng, J.F., Zho, J.S., Lin, X.F., Ding, C.Y., Wu, C., and Blot, W.J. Gender differences in hematopoietic and lympboproliferative disorders and othe cancer risk by major occupational group among workers exposed to benzene in China. I. Descriptive Findings. J.Occup.Med. 360: 875-881, 1994. BP-00011610 - - - - -----~-- PETROLEUM WORKER STIJDIES AND BENZENE RISKASSESSMENT Robert Scbnatter I Most epidemiologic studies involving benzene-exposed workers can be broadly classified as those that involve-workers who used benzene-based solvents, and those that involve workers exposed to benzene from petroleum products. Risk assessments have focused upon the former (e.g., Rinsky et al., 1987), and have not directly used the results ofthe latter group of studies. However, these studies reflect exposure scenarios that are more like those ofthe general population. The focus ofthis presentation will be on the latter group ofstudies, and their potential use in comprehensive benzene risk assessments. Workers in petroleum studies cover a broad time period stretching from the 1920's and earlier to the 1980's. The benzene content ofgasoline has always varied (Gamer and Evans, 1932), but probably rose somewhat following the introduction of catalytic cmcking technology in the late 1930's;-From that time forward. the benzene content of gasoline has typically been in the range of 1-5% with rarer instances ofhigher percentages noted. Worker benzene exposure from refinery operations probably increased from the 1920's to 1960's and then decreased over time due to fugitive emission control and other exposure control initiatives. Benzene exposures in distribution and retail operations also probably increased from the 1920's to 1950's, but then remained relatively stable thereafter. Typical eight hour exposure concentrations in distribution and retail operations average less than one part per million, although exposures typically reach 2-3 ppm for briefer (e.g., 30-120 minute) periods. These exposures a.--e one to two orders of magnitude lower than those present in workers exposed to benzene through past solvent use (e.g., Rinksy et al., 1987). Several similarly designed cohort studies have been reported in petroleum workers. Unfortunately, benzene exposure has not been directly measured for each study subject, although reasonable bounds can certainly be placed on benzene exposure in the aggregate study populations. Three nested case control studies in petroleum workers have either been conducted or are nearing completion. These studies, also similarly designed. have conducted thorough benzene exposure assessments for each worker in the study populations. The first of these studies involved 29 Canadian lymphohematopoietic cancer cases (14 leukemias, seven multiple myelomas, and eight non-Hodgkin lymphomas) and 115 matched controls (Schnatter et al., 1996). Average daily benzene concentrations ranged from 0.01 to 6.2 ppm (Armstrong et al., 1996). Several exposure metrics.and categories were analyzed for the three types ofLH cancer. Limited information on confounders (e.g., smoking) was available. In general, there was no consistent dose response relationship found for any of the three types ofLH cancer..Benzene exposure duration showed a slightly stronger association with leukemia than other metrics such as cumulative-exposure, mean intensity, and the number of years above either 0.5 or l ppm benzene exposure. The results are consistent with either a lack ofeffect due to benzene, or inadequate power to detect an effect as high as two-fold. A second, larger study in U.K. workers was modelled after the Canadian study (Rushton and Romaniuk, 1997). This-study involved 90 cases (all were leukemia) and 354 matched controls. Cases were identified and classified through both death certificates and cancer registryinformation with respect to leukemia cell type. For lymphatic leukemias as well as chronic myelogenous leukemia (CML), there were no consistent dose response trends for various exposuremetrics. Results for acute myeloid leukemia (AML) were more difficult to interpret. There was a BP-00011611 suggestive dose response trend between AML and for categorical, but not continuous, exposure metrics. The dose response trend became slightly stronger wlien incorporating a five or ten year lag between exposure and outcome, or when incorporating peak exposure metrics. However, in _sensitivity analyses, the dose response trend between AMI. and categorical metrics became weaker, and indeed disappeared when only the strongest exposure estimating information was used (i.e., complete work history records, and exposures given higher confidence scores). The authors interpreted the study as weakly suggestive of an effect, but noted that because the study lacked intemaLconsistency, no firm conclusions could be made. A third study is underway in Australia. It is due to be completed in 2000 and will consist of approximately 80 cases ofLH cancer and matched controls. Again, benzene exposure estimates will be quantified, and regression analyses will be performed. An advantage ofthe Australian study is that relatively good information on smoking will be available, and a greater fraction of cases and controls were employed in more recent time periods, perhaps allowing more certainty in resulting benzene exposure estimates. In risk assessment jargon, these studies do not demonstrate that benzene is a "hazard" at typical workplace concentrations. Thus, although it is mathematically possible, it would not be appropriate to calculate unit risk estimates for benzene's potency. However, the studies can- be used to provide some reassurance that _typical benzene exposure concentrations experienced by petroleum workers are not causing large increases ofAML or other leukemias. Certainly, they do not provide any justification for lowering occupational workplace limits below 1 ppm. The petroleum cohort studies also have some role in benzene risk assessments. Their strengths and weaknesses are opposite those ofthe case control studies. The case control studies have measured exposure as-precisely as possible; the cohort studies have not. Small numbers limit the case control studies, but the aggregate cohort population is very large. However, both sets of studies do have a role in comprehensive benzene risk assessments. Wong and Raabe (1995) have summarized cell-specific -leukemia mortality in U.S. ana U.K. petroleum worker studies. The analysis covered 19 studies, more than 208,000 workers, and more than 4.5 riilllion person years of observation from 1937 through 1989. There were 327 leukemia deaths that were classified by cell type. Most of the aggregate population consisted of refinery workers, with smaller groups of distribution, marine, and upstream workers also included. While no formal analysis of heterogeneity was performed, the authors thought it was likely that withincohort exposure variation would be comparable, ifnot greater than, between-cohort exposure variation. -Overall SMR's and 95% confidence intervals for the four major leukemia cell types were as follows: AML- 0.96 (0.81- 1.14), CML- 0.82 (0.62- 1.08), ALL- 1.22 (0.83- 1.71), and CLL- 0.79 (0.62- 1.00). While these results also do not suggest a hazard from benzene at these exposure concentrations, benzene exposures were not directly measured in.any ofthe petroleum workercohort studies. However, reasonable estimates ofexposure can be made using comprehensive industry-wide surveys ofbenzene exposure. Runion and Scott (1985) reported over 14,000 benzene samples submitted by over 30 companies for refmery operations. For a relatively recent time period {1978-83), exposures averaged 0.22 ppm. Using another-extensive-data set from Tsai (1983), which covers 737 samples from a somewhat earlier period (1973-82), a higher mean of 0.65 ppm can be estimated. Some ofthe difference in these two numbers is probably attributed to advances in fugitive emission control in the 1970's. The average from both data sets yields an BP-00011612 estimate of 0.44 ppm, which may still be somewhat ofan underestimate for a population that extends back into the 1930's. However, there are undoubtedly some workers in the study population who were exposed to only background benzene levels. Thus, an estimate of0.44 ppm for the average exposure in the_population appears reasonable. Hertz-Piccioto (1995) summarized how epidemiologic data can be applied in quantitative risk assessment. For a study or studies to be used "as a basis for extrapolation", or'as a basis for potency calculations, she suggests that four criteria be met: (a) a moderate to strong positive association be present, (b) strong biases are unlikely, (c) confounding is limited, and (d) quantification of exposure be linked to individuals. For the case control studies (a) is lacking, while for the cohort studies, both (a) and (d) are lacking. For a study to be used to "check plausibility", sh~ suggests-a moderate to strong positive association is often not required. The studies, however, should at least partially be able to rule out confounding and bias. In addition, some quantification ofexposure is needed "even ifbased on data external to the study site". Clearly, both the case control studies and the cohort studies in petroleum workers would qualifY for use as "plausibility checks". Because existing.unit risk estimates for benzene exposure are based on a cohort study, the petroleum cohort studies are more amenable for use as plausibility checks. The Wong and Raabe ( 1995)_ aggregate population was exposed to benzene in an occupational scenario. Unit risk estimates have also been developed in workers who were exposed to benzene in the occupational environment. Frequently, a fiJ'St step in converting study results from these scenarios to the general population is to apply dosimetric adjustments, taking into account the number ofdays per year, hours per day, and number ofyears exposed. Ifunit risk estimates (of6 permillion) are applied to the Wong and Raabe population, and dosimetric adjustments are made, 163 excess deaths due to AML would be predicted. These deaths along with the 155..expected deaths would predict 318 total deaths due to AMI.. in the Wong and Raabe population. However, the Wong and Raabe confidence limits can be used to calculate that the study is consistent with from 125 to 175 total AMI.. deaths. The 318 deaths falls well outside of this range, and therefore, is inconsistent with the results of Wong and-Raabe (1995). There are a few ways to resolve this inconsistency. The first would be to reject the previous unit risk estimates, recognize the lack ofeffect in petroleum workers, and apply appropriate uncertainty factors applicable to environmental, rather than occupational, exposure scenarios. There is some scientific justification for this approach, as benzene exposure in the petroleum workers is more analogous to benzene exposure in the general population, the level of benzene exposure in petroleum workers is cl-oser to general population exposures, necessitating less extrapolation. A second approach, which-is more of a compromise, would be to calculate a unit risk estimate-Consistent with the upper confidence limit of the Wong and Raabe study. A third approach would be to ignore the inconsistency, and continue to use the unit risk factor for solventexposed populations. This may be the least scientifically supportable approach. In summary, the existing studies in petroleum workers qualify for use as plausibility checks on benzene unit risk estimates. When the studies are used in this manner, they indicate that existing unit risk estimates ofbenzene's potency are not-consistent with this body ofliterature. Therefore, procedures which integrate the petroleum worker studies and benzene solvent.,user studies are indicated. BP-00011613 Biomarkers in the Molecular Epidemiology ofBenzene-Exposed Workers Martyn T. Smith1 and Nathaniel Rothman2 1 Division ofEnvironmental Health Sciences, School of Public Health;University of-California. Berkeley, CA 94720-7360 and 2 Division ofCancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892 Introduction Biomarkers can be classified into three categories: biomarkers ofexposure, susceptibility, and early effect. Along with colleagues from the National Cancer Institute, the Chinese Academy of Preventive Medicine in Beijing, the Shanghai Anti-epidemic Center, the University ofNorth Carolina and other institutions in the United States, we have applied various biomarker methods to samples obtained from workers exposed to high levels ofbenzene. The goal of these studies is to develop and validate 1) biomarkers ofexposure to benzene, such as albumin or hemoglobin adducts; 2) molecular markers ofsusceptibility to benzene, such as inherited polymorphisms in enzymes involved in the metabolism ofbenzene; and 3) biomarkers ofthe early effects ofbenzene, including hematotoxicity (complete blood cell counts), gene mutations (glycophorin A), and chromosome aberrations detected by fluorescence in situ hybridization (FISH) , G.:banding and the micronucleus assay. An introduction to these studies has been presented previously ADDINENRfu (Rothman, et al., 1996a; Rothman, eta!., 1996b; Smith and Rothman, 1998; Smith and Zhang, 1998), and only those fmdings pertaining to biomarkers ofexposure and early effects will be discussed here. Sample Collection and Exposure Assessment Biological samples were collected from 44 healthy workers currently exposed to benzene with minimal exposure to toluene and other aromatic solvents in Shanghai, China in October, 1992. The same number ofhealthy controls without current or previous occupational exposure to benzene were enrolled from factories in the same geographic area. Controls were frequency-matched by gender and age (5 year intervals). Exclusion criteria for all suojects were history of cancer, therapeutic radiation. chemotherapy, or current pregnancy. Each subject was administered_a questionnaire by a trained interviewer. Data collected included age, gender,-current and lifelong tobacco use~ current alcohol consumption. medical history and work history. Height and weight of each subject were measured and peripheral-blood was obtained by phleboromy. Individual exposure was_monitored by organic vapor passive dosimetry badges (3M #3500, St. Paul, Minnesota), which were worn by each worker for a full workshift on 5 separate days during the one- to two-week period prior to phlebotomy. An eight-hour..time weighted average (TWA) exposure was calculated for benzene as the geometric mean ofthe five air measurements. Cumulative exposure to benzene was calculated by multiplying historical time-specific exposure estimates by the duration wor-ked. All exposure assessment was performed blinded-i.th respect to the biomarker analysis. Biomarkei"S of Exposure f The median benzene air level among the exposed workers was 31 ppm as an 8 hour--rwA (range: 1-328 ppm). Air monitoring data were confirmed by measures ofurinarybenzene metabolites; phenol, muconic acid, -catechol and hydroquinone showed strong, positive correlations with air benzene levels, and were substantially higher in exposed workers compared to controls ADDIN ENRfu (Rothman, eta!., 1998). In addition, current be:m:ne air levels were inversely correlated with the absolute lymphocyte count among exposed workers, consistent with BP-00011614 previous reports that lymphocytes are particularly sensitive to benzene ADDIN ENRfu (Rothman, et al. 1996a; 1996b). The initial metabolite ofbenzene, benzene oxide (BO), reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts {BOHb and BO-Alb}, which are presumed to be specific biomarkers ofexposure to benzene. With Dr. Stephen Rappaport's lab in Chapel Hill we analyzed BO-Hb in 43 ofthe exposed workers and 42 unexposed controls and BO-Alb in a subsample consisting of 19 workers and 19 controls ADDIN ENRfu -(eowell-O'Connell, et al., 1998). The adducts were analyzed by GC-MS following reaction ofthe protein with tritluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to< 31 ppm(n =21) and> 31 ppm (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol!g globin, respectively (correlation with exposure: Spearman r = 0.67, p < 0.0001). These results represent the first observation in humans-that-BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these3 groups were 103 (n = 19), 351 (n = 7)and 2010 (n = 12)pmol/g =Alb, respectively, also reflecting a significant correlatioo with exposure (Spearman r 0.90, p < 0.0001). These results clearly affirm the use ofbothHb and Alb adducts ofBO as biomarkers of exposure to high levels ofbenzene. Biomarkers ofEarly Effect from Benzene Exposure A potential method ofpredicting who is most at risk for benzene-induced leukemia is to determine the extent ofthe genetic damage it produces in exposed individuals, using biomarkers ofearly effect. One means ofassessing genetic damage is to measure mutations in specific genes, such as glycophorin A (GPA) ADDIN ENRfu (Compton, et al., 1991; Jensen and Bigbee, 1996). An increased-level of"gene-duplicating" mutations in GPA was found in the benzene-exposed workers ADDIN ENRfu n(;Rothman, et aL, 1995). Interestingly, this increased-mutation frequency was correlated with cumulative exposure to benzene. Since cumulative exposure to benzene may correlate best with leukemia risk, the GPA assay appears to have potential as a biomarker ofearly biological effect for benzene and other leukemogens. The GPA assay has drawbacks, however. First, it is relatively insensitive: high benzene exposure (mean time-weighted average (TWA) at 72 ppm) only elevated the combined mutant frequency from 16.3 to 23.0 per million, a 41% increase ADDIN ENRfu (Rothman, et al., 1995). Second, it can only be performed on GPA heterozygous (type MN) individuals, who statistically constitute only 50% of -any given population under study. Thus, although the GPA assay can provide important mechanistic information, it may not be an ideal biomarker ofearly effect. The most common means of detecting genetic damage has traditionally been conventional cytogenetics. Numerous publications have demonstrated a clear association between benzene exposure and increased levels ofchromosome aberrations in peripheral blood cells. Since chromosome aberrations in peripheral blood lymphocytes-have been shown to be associated with -increased risk for overall cancer incidence ADDIN ENRfu (Hagmar, et al., 1994), especially for increased mortality from hematological malignancies ADDIN ENRfu ..(Bonassi, et al., 1995), it is possible that specific chromosome aberrations may-provide even better markers of future leukemia risk.Specific Chromosome Aberrations in Leukemia Specific chromosome aberrations are the hallmark of human leukemia ADDIN ENRfu (Hagemeijer and GroSYeld, 1996). Aneuploidy, the Joss or gain of specific chromosomes in AML and MDS (such as trisomy 8 and monosomy 5 and 7), is commonly observed as specific chr-omosome translocations, inversions and deletions (e.g. t(8;21), t(9;22), inv(16) and del(5q)) ADDIN ENRfu (Hagemeijer and Grosveld, 1996). The loss of chromosomes 5 and 7 and their Ion~ deletions are the two most common changes in therapy-related-AML and MDS (tAMI..._and t-MDS), especially among patients previously treated with alkylating agents ADDEN ENRfu n(Pedersen-Bjergaard, et al., 1995). Treatment with topoisomerase II inhibitors is BP-00011615 associated with balanced chromosomeaberrations such as t(8;21) and t(llq23) in t-AML ADDIN ENR.fu (Pedersen-Bjer_gaard, et al., 1995). These specific chromosome aberrations are also more common among leukemia patients with previous-exposure to chemical solvents (including chronic exposure to benzene, insecticides, petroleum, etc.) ADDIN ENR.fu (Crane, et al., 1996; Mitelman, et al., 1981). Detection ofSpecific Chromosome Aberrations by FISHWe have applied FISH to determine the presence of specific chromosome aberrations in the lymphocytes ofotherwise healthy workers exposed to benzene and matched controls. Initially, we studied hyperdiploidy levels ofchromosome 9 in interphase cells-because trisomy 9 has been observed in benzene-poisoned patients ADDIN ENRfu (Erdogan and Aksoy, 1973; Forni and Moreo, 1967) and benzene metabolites induce hyperdiploidy ofthis chromosome in cultured lymphocytes in vitro ADDIN ENRfu (Eastmond, et al., 1994; Zhang, et al., 1994). High benzene exposure waHhown to increase hyperdiploidy o[chromosome 9 in the lymphocytes of otherwise healthy workers, with trisomy 9 being the most prevalent form ADDIN ENRfu (Zhang, et al., 1996). We have gone on--to use interphase cytogenetics to study the hyperdiploidy ofchromosomes 7 and 8 and these findings will be published shortly. Interphase cytogenetics cannot be used, however, to detect monosomy or rare translocations because of artifacts related to probe overlap ADDIN ENRfu ..(Eastmond and Pinkel, 1990). Monosomy ofchromosomes 5 and 7 and translocation (8;21) are among the most common aberrations observed in AML. We therefore used chromosome-painting and region-specific fluorescent probes to examine AML-specific aberrations, including -5, -7, del (Sq31 ), del {7q2234) and t(8;21), in metaphase spreads prepared from the lymphocytes ofworkers exposed to benzene and matched controls ADDIN ENRfu (Smith, et al., 1998; Zhang, et a!., 1998a). We painted chromosomes 8 and 21 in lymphocyte metaphases from the 44-workers exposed to benzene and 44 matched controls. To examine dose-response reiationships the workers were divided into 2 groups at the median e.'Cposure level, a lower-exposed group(= 31 ppm, n = 21) and a higher-exposed group (> 31 ppm, n = 22). Benzene exposure was associated with significant increases in hyperdiploidy ofchromosome 8 (1.2, 1.5, 2.4 per 100 metaphases; Ptrend < 0.0001) and 21 (0.9, 1.1, 1.9; Ptrend < 0.0001). Translocations between chromosomes 8 and 21 were increased up to 15-fold in highly exposed workers (0.01, 0.04, 0.16; Ptrend < 0.0001). In one highly exposed individual these translocations were-reciprocal and were detectable by reversetranscriptase PCR ADDIN ENRfu (Smith, et a!., 1998). These data indicate a potential role for t(8;21) in benzene-induced leukemogenesis and are consistent with the hypothesis that detection of specific chromosome aberrations may be a powerful approach to identify populations at increased risk ofleukemia from benzene exposurt'- We also used a novel FISH procedure to determine ifspecific aberrations in chromosomes l, 5 and 7 occured at an elevated rate in metaphase spreads prepared from the lymphocytes ofthe same benzene-exposed Chinese workers ADDIN ENRfu (Z-hang, et al., 1998a}. We found that benzene exposure was associated with increases in the rates of monosomy 5 and 7 but not monosomy 1 (p < 0.001; < 0.0001; and 0.94,-respectively) and with-increases in-trisomy aad tetrasomy frequencies-Of all three chromosomes. Long arm deletion ofchromosomes 5 and 7 was =increasedttl a dose-dependent fashion (p 0.014 and< 0.0001) up to 3.5-fold in the exposed workers. These results demonstrate that leukemia-specific changes in chr-omosomes 5 and 7 can be detected by FISH in the peripheral blood of otherwise healthy benzene-exposed workers. We have also shown that benzene metabolites induce these same changes in vitro ADDIN ENRfu (Zhang, et al., 1998b). Taken together all of these data obtained using FISH suggest that aberr.ations in chromosomes 5, 7, 8 and 21- may be a useful ffiomarker of early biological effect for --benzene exposure. Indeed, Dr. Luoping Zhang ofDr. Smith's laboratory harrecently devised a FISH procedure to examine the leukemia-related changes in all4 ofthese chromosomes BP-00011616 simultaneously. Studies are being planned to apply these methods in workers exposed to a broad range ofbenzene concentrations. In addition, we will explore the impact ofinterindividual variation in genes that activate and detoxifY benzene and its metabolites on these outcomes, following-up our previous report that variation in CYP2El and NQOI influence the risk ofbenzene bematotoxicity ADDIN ENRfu (Rothman. et al., 1997). AcknowledgementsWe are indebted to the study subjects and the staffofthe Chinese Academy ofPreventive Medicine, Beijing. and the Shanghai Hygiene and Anti-Epidemic Center, Shanghai, China for their assistance and participation in this study. These studies were supported by funds from the National Cancer Institute and National Institute of Environmental Health Sciences (Grants ROl ES0672l, P42 ES04705 and P30 ES01896) and the California Environmental Protection Agency. The following individuals have contributed greatly to these studies: Luoping Zhang, Nina Titeoko-Holland, Yunxia Wang, Prema Kolachana, Joseph Wiemels, Kathleen Meyer (School of Public Health, University_of California, Berkeley, CA 94720); Richard Hayes, Mustafa Dosemeci (Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892); Songnian Yirr, Guilan Li, Liqiang Xi, Weihong Guo (Chinese Academy ofPreventive Medicine, Institute ofOccupational Medicine, Beijing, China); Stephen Rappaport, Karen Yeowell O'Connell, Suramya Waidyanatha (School of Public Health, University ofNorth Carolina at Chapel Hill, Chapel Hill NC 27599-7400); YaoZhu Wang, Lu Wei (Shanghai Hygiene and Anti-Epidemic Center, Shanghai, China); and Robert Haas (California EPA). References ADDIN ENBbu Bonassi, S., A. Abbondandolo, L. Camurri, L. Dal Pra, M. De Ferrari, F. Degrassi, A. Forni, L. Lamberti, C. Lando, P. Padovani, I. Sbrana, D. Vecchio and R. Puntoni (1995) Are chromosome aberrations in circulating lymphocytes predictive offuture cancer onset in humans? Preliminary results of an Italian cohort study, Cancer Genet Cytogenet, 79, 133S.Compton, P.J., K. Hooper and M.T. Smith (1991) Human somatic mutation assays as biomarkers ofcarcinogenesis, Environ Health Perspect, 94, 135-41. Crane, M., S. Strom, S:-Halabi, E. Berman, J. Fueger, M. Spitz and M. Keating (1996) Correlation between selected environmental exposures and karyotype in acute myelocytic leukemia, Cancer Epidemiology, Biomarkers & Prevention, 5,639-44. Eastmond, D.A. and D. Pinkel (1990) Detection of aneuploidy and aneuploidy-inducing agents in human l}'IIlphocytes using fluorescence in "Situ hybridization with chromosome-specific DNA probes, Mutat Res, 234,303-18. Eastmond, D.A., D.S. Rupa and L.S. Hasegawa (1994) Detection of hyperdiploidy and chromosome breakage in interphase human lymphocyteS-following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes, Mutat Res, 322, 9-20. Erdogan, G. and M. Aksoy (1973) Cytogenetic studies in thirteen patients with pancytopenia and leukaemia associated with long-term e~posure to benzene, New Istanbul Contnb Clin Sci, 10, 23047__ Forni, A. and L. Moreo (1967) Cytogenetic studies in a case of benzene leukaemia, Eur J Cancer, 3,251-5. Hagemeijer, A. and G. Grosveld (1996) Molecular cytogenetic!Ulfleukemia, in: E. Henderson, T. Listeund M. Greaves (Eds.), Leukemia, Saunders, Philadelphia. Hagmar, L., A. Bragger, I.L. Hansteen, S. Heim, B. Hogstedt, L. Knudsen, B. Lambert, K. Linnainmaa, F. Mitelman, I.Nordenson, C. Reuterwall, S. Salomaa, S. Skerfving and M. Sorsa ( 1994) Cancer risk in humans predicted by increased levels-of chromosomal aberrations in BP-00011617 lymphocytes: Nordic study group on the health risk ofchromosome damage, Cancer Res, 54, 2919-22. I Jensen. R.H. and W .L. Bigbee (1996) Direct immunofluorescence labeling provides an improved method for the glycophorin A somatic cell mutation assay, Cytometry, 23, 337-43. Mitelman, F., P.G. Nilsson, L. Brandt, G. Alimena, R. Gastaldi and B. Dallapiccola (1981) Chromosome pattern, occupation, and clinical features in patients with acute nonlymphocytic leukemia, Cancer Genet Cytogenet, 4, 197-214. Pedersen-Bjergaard, J., M. Pedersen, D. Roulston and P. Philip (1995) Different genetic pathways in leukemogenesis for patients presenting with therapy-related myelodysplasia and therapy-related acute myeloid leukemia, Blood, 86, 3542-52. Rothman, N., W.E. Bechtold, S. Yin, M. Dosemeci, G. Li, Y. Wang, W.C. Griffith, M.T. Smitn and R.B. Hayes (1998) Urinary excretion of phenol, hydroquinone, and muconic acid by workers occupationally exposed to benzene, Occup and Environ Med, (in press). Rothman, N., R. Haas, R.B. Hayes, G.-L. Li, J. Wiemels, S. Campleman, P.J.E. Quintana, L.-J. Xi, M. Dosimeci, N. Titenko-Holland, K.B. Meyer, W. Lu, L.P. Zhang, W. Bechtold, Y.-Z. Wang, P. Kolachana, S.-N. Yin, W. Blot and M.T. Smith (1995) Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin-A locus, Proceedings of the National Academy of Sciences USA, 92,4069-4073. Rothman, N., G.-L. Li, M. Dosimeci, W.E. Bechtold, G.E. Marti, Y.-Z. Wang, M. Linet, L.-Q. Xi, W. Lu, M.T. Smith, N. Titenko-Holland, L. Zhang, W. Blot, S. Yin and R. Hayes (1996a) Hematotoxicity among Chinese workers heavily exposed to benzene, Am J Ind Med, 29, 236-246. Rothman, N., M.T. Smith, R.B. Hayes, G.-L. Li, R.D. Irons, M. Dosimeci, R. Haas, W.S. Stillman, M. Linet, L.-Q-. Xi, W.E. Bechtold, J. Wiemels, S. Campleman, L. Zhang, P.J.E. Quintana.-N~Titenko-Holland, W.L. Wang, P. Kolachana, K..B. Meyer and S. Yin (1996b) An epidemiologic study ofearly biological effects of benzene in Chinese workers, Environmental Health Perspectives, 106 Suppl 6, 1365-70. Rothman, N., M.T. Smith, R.B. Hayes, R.D. Traver, B.-A. Hoener, S. Campleman, G.-L. Li, M. Dosemeci, M. Linet, L. Zhang, L. Xi, S. Wacholder, L. Wei, K..B. Meyer, N. Titenko-Holland, IT. Stewart, S.-N. Yin and D. Ross (1997) Benzene poisoning, a risk factor for hematologic malignancy, is associated with the NQOl 609C->T mutation and rapid fractional excretion of chlorzoxazone., Cancer Research, 57, 2839-2842. Smith, M.T. and N. Rothman (1998) Biomarkers ofleukemia risk from benzene, in;_M.L. Mendelsolm, L.C. Mohr and J.P. Peeters (Eds.), Medical and Workplace Applications of Biomarkers, Joseph Henry Press, National Academy of Sciences, Washington, D.C., pp. 355-374. Smith, M.T. and L. Zhang (1998) Biomarkers of leukemia risk: Benzene as a-model, Environ Health Perspect, 106 Suppl4, 937-946. Smith, M.T., L. Zhang, Y. Wang, R.B. Hayes, G. Li, J. Weimels, M. Dosemeci, N. TittenkoHolland, L. Xi, P:-Kolachana, S. Yin and N. Rothman (1998) Increased translocations and aneusomy-in chromosomes 8 and 21 among workers exposed to benzene, Cancer Research, 58, 2176-2181. Yeowell-O'Connell, K.., N. Rothman, M.T. Smith, R.B. Hayes, G. Li, S. Waidyanatha, M. Dosemeci, L. Zhang, S. Yin, N.T. Holland and S.M. Rappaport (1998)Hemoglobin and albumin adducts ofbenzene oxide among workers exposed to high levels ofbenzene, Carcinogenesis, 19, 1565-1571. Zhang, L., P. Venkatesh, M.L Creek and M.T. Smith (1994) Detection of 1,2,4-benzenetriol induced aneuploidy and microtubule_disruptionhy fluorescence in situ_hybridization and immunocytochemistry, Mutat Res, 320,315-27. BP-00011618 Zhang, L., N. Rothman, Y. Wang, R.B. Hayes, W. Bechtold, P. Venkatesh, S. Yin. Y. Wang, M. Dosemeci, G. Li, W. Lu and M.T. Smith (1996) Interphase cytogenetics ofworkers exposed to benzene, Environ Health Perspect, 104 Suppl6, 1325-9. Zhang, L., N. Rothman, Y. Wang, R.B. Hayes, G. Li, M. Dosemeci, S. Yin, P. Kolachana, N. Titenko:Holland and M.T. Smith (1998a) Increased aneusomy and long ann deletion of chromosomes 5 and 7 in the lymphocytes ofChinese workers exposed to benzene, Carcinogenesis, 19, (11) 1955-1961. Zhang; L., L. Wang, N. Shang and M.T. Smith (1998b) Benzene metabolites induce the loss and long ann deletion ofchromosomes 5 and 7 in human blood cells, Leukemia Res, 22, I05113. Please address all correspondence to :Martyn T. Smith, Ph.D. Professor ofToxicology Head, Division ofEnvironmental Health Sciences School of Public Health 216 Earl Warren Hall University of California Berkeley, California 94720-7360 (510) 642-8770 tel/ (510) 642-0427 fax email: martynts@uclink4.berkeley.edu web site: http://ehs.sph.berkeley.edu/msmith Abstract for" Benzene: State-efthe Science Workshop", Ottawa, Canada, December 16-17, 1998 BP-00011619 ..... 1- THE HEALTH EFFECTS INSTITUTE'S BIOMARKER RESEARCH IN CHINA. Martha E. Richmond, Health Effects institute, Cambridge, MA. Exposu.t:e-to benzene is associated with the development of several blood disorders, including aplastic anemia, pancytopenia, chromosomal aberrations, and acute non-lymphocytic leukemia (Aksoy, 1989; Crump, 1994; Forni, 1996; Hayeset al., 1997; Rinsky etal., 1987; Rothman et al., 1996). Much ofthe evidence establishing the relationship between benzene exposwe and the development ofthese disorders bas come from occupational health studies where exposures generally range from 2 to 100 ppm. However, the magnitude of risk for adverse health effects at ambient levels of exposure, which range from <1 to 3 ppb, remains to be addressed. In particular, information about the mechanism of leukemogenesis and the development of aplastic anemia in human populations is incomplete. There is clearly a threshold for benzene-induced aplastic anemia, a condition_characterized by a decrease in the number of circulating red and white blood cells and platelets accompanied by almost complete replacement of many bone marrow cell cypes by scar tissue. However, it is still unknown whether blood cell toxicity, manifested as aplastic anemia, is a necessary step in-the development of leukemia. Also limiting an understanding ofleukemia is a significant knowledge gap about the cellular mechanisms for benzene-induced leukemia, especially the steps required in the early stages. The existence ofknowledge gaps is an important public health issue, since there are a number ofenvironmental sources for benzene exposure, ranging from main and side-stream tobacco smoke to certain food stuffs (Wallace, 1996). Benzene is ~resent in gasoline, ranging from somewhat less than I% in the United States (AAMA, 1995) up to approximately 5% in some other countries. Benzene is named as one ofthe mobile source air toxics in the Clean Air Act Amendments of1990. In response to provisions -of the Clean Air Act Amendments the Health Effects Institute (HEI) held a workshop in 1992 which formed t.'le basis ofa publication issued in 1993, "Research Priorities for Mobile Air Taxies". This document identified research needs related to benzene and several other mobile source air toxics. Specific research needs for benzene included the need to: (I) develop sensitive methocb-to measure metabolites from low-level exposure to benzene; (2) identifY markers of exposure, uptake. and metabolism and validate these markers_in human-populations; (3) develop dose-response information on biomarkers so.as to evaluate the relationship ofexposure levels to levels-of metabolites and genetic changes within and across species; (4) make progress toward elucidating the mechanism ofbenzenc; toxicity. As a flrst step in addressing several of these research needs, HEI funded a study looking at methods to measure metabolitesJrom low-level exposure to benzene. HEI also funaed several biomarker development studies. Two of these studies were to develop biomarkers of exposure BP-00011620 and dose, and the third to develop a -biomarker of effect: C S-phenylcysteine in albumin as a benzene biomarker, a marker of exposure developed by Dr. William Bechtold of the Lovelace Respiratory Research Institute C Development of liquid chromatographic-electrospray ionization-mass spectrometric methods for determination ofurinary metabolites ofbenzene, markers ofexposure and dose developed by Dr. Asseih Melikian of the American Health Foundation. Specific metabolites include t,t-muconic acid, S-phenylmercapturic acid, 1,2,4-trihydroxybenzene, hydroquinone, and catechol. C Characterization-and mechanisms of chromosomal alterations induced by benzene in mice and humans. This study includes development of methods to measure chromosomal alterations, biomarkers ofeffect, using fluorescent in situ hybridization (FISH) developed by Dr. David Eastmond of the University of California at Riverside. The original developmentalwork ofthe biomarkers was done in animal systems. However, the goal ofthe studies was to develop markers that -would be useful in humans exposed to ambient levels ofbenzene. To achieve this second goal, HEI issued a request for qualifications (RFQ) in 1995 to establish a collaborative relationship between investigators with access to benzene-exposed populations and investigators who had developed the biomarkers in animal systems. The relationship would result in a proposal for a transitional epidemiology study to validate the biomarkers in human populations. As the result of this process, HEI is now funding a study under the overall direction of Dr. Qingshan Qu of the Institute ofEnvironrnental Medicine at New Y-Ork University. This study is being conducted in collaboration-with investigators from the Chinese Academy ofPreventive Medicine under the direction of Dr. Guilan Li. Dr. Qu's study involves sub-sets-of a cohort of occupationally exposed benzene workers and unexposed workers identified by the National Cancer Institute (Yin, et al.,l994). The study is divided into two phases. The focus of the first phase is to determine sensitivity, reproducibility, and the time course of elimination of biomarkers of exposure and dose. The focus ofthe second phase is to develop dose-response profiles of all markers determined to be appropriate based on results ofthe-first phase._ Phase I is now nearing completion. Phase II is under way and expected to end late in 1999. A total of25 exposed subjects and 25 control subjects were enrolled in Phase I. Benzene-exposed workers were employed in glue and shoe manufacturing facilities in Tienjing Province, -china. In multiple exposure assessments, benzene exposures ranged from 10 ppm to 150 ppm with a median ranging from 19-26 ppm depending on the occasion of BP-00011621 exposure measurement. There were also small amounts ofxylene-and toluene present in the glue and shoe manufacturing facilities (approximately 5-1 0% of the benzene concentration). Unexposed controls were workers employed in a food processing facility in the same regionof China. Exposure ofcontrols was below the level ofdetection in personal and area sampling methods. Current exposure ofwalkers was determined by personal samplers, and historical exposure profiles for all subjects bas been determined from work records. In Phase I. all enrolled subjects wore duplicate personal samplers. Blood and urine samples, taken from all enrolled subjects were distributed to laboratories-ofDrs. Bechtold, Eastmond.and Melikian for -analysis ofbiomarkers. To gain more information about the metabolite or mixture-of metabolites associated with short-term exposure, urine samples collected before and after work shifts were analyzed for several markers of exposure. A-time-course study ofthe urinary biomarkers was also conducted to determine the kinetics ofelimination. Markers included the benzene-specific marker, S-phenylmercapturic acid, as well as t,t-muconic acid, benzene triol, hydroquinone and phenol. While some of these markers are not specific to benzene, several-are thought to be related to benzene toxicity (Gut, et at., 1996; Smith, 1996; Witz et at, 1996), and information abaut their levels and patterns, especially at the lower levels ofexposure, may provide important information about benzene metabolism. Blood--samples were analyzed for S-phenylcysteine adducts of albumin as a biomarker of longer term exposure. Blood samples were also analyzed for chromosomal breakage and hyperdiploidy of interphase lymphocytes and Go granulocytes using FISH. Metaphase chromosomes from lymphocytes have also been analyzed for chromosomal aberrations of all chromosomes using more conventional techniques. Routine clinical analyses of all blood samples were conducted for hemoglobin, red blood cells, red blood cell morphology and white blood u:lls. A differential analysis of-ail white blood cells was also conducted. Data analysis will correlate exposure levels with the various markers of exposure and effect. Data anal}'Sis will also correlate a number of end points as a means ofb...aginning to establish links between potential toxic effects (as determined by chromosomal aberrations and bloed count data) and markers ofexposure;- Final data analysis ofPhase I results is nearing completion. At the present time, the dose-response-study (Phase II) is underway. A total of 130 subjects are enrolled in this phase. This includes 25 control subjectumd 105 benzene-exposed subjects employed in manufacture ofsporting_goods. All subjects are from-the Tienjing Province area--of China. Benzene exposures range from< 1ppm to >15 ppm. Subjects are grouped into four exposure levels as follows: <1 ppm, 40 subjects; 1.1 to 5 ppm, 30 subjects; 5.1 to 15 ppm, 25 subjects, and >15 ppm 10 subjects. Based on results from Phase I, aspects ofthe end points-and study design ofPhase ll have been modified. This includes the personal sampling strategy. Because of the excellent agreement BP-00011622 found in the analysis ofduplicate personal samplers taken from Phase I subjects, a smaller number of the subjects enrolled in Phase II (10%) wore duplicate samplers. Personal exposure monitoring was conducted twice: once prior to obtainingbiological samples; and once during the biological sampling. Monitors were analyzed both in China and at New York University. Because analysis of several selected chromosomes using FISH technology did not discriminate between exposed and unexposed workers, more conventional techniques, also used in Phase I, are being used to analyze lymphocytes for chromosomal aberrations, and all chromosomes will be examined. Phase ll will also involve reconstruction ofhistorical exposure profiles for all participating subjects, using work records. Results ofthe biomarker validation study are expected to address several of the goals originally identified in HErs benzene resear-ch program. Information should indicate whether the markers ofshort-term (urinary metabolites) and longer-term (albumin adducts) exposure are sufficiently sensitive to be used as markers ofexposure to ambient environmental levels ofbenzene. Dose-response information for both biomarkers ofexposure and biomarkers ofeffect will be developed and analyzed for possible relationships. This may provide additional infonnation about relationships between exposure, metabolism, and genetic change. Finally, it may be possible,-in a population-based study to identify certain individualswho may be more susceptible to toxic effects ofbenzene, as determined by chromosomal aberrations and blood count measurements. REFERENCES: Aksoy, M. (1989). Hematotoxicity and carcinogenicity ofbenzene. Environ Health Perspect 82: 193-191. American Automobile Manufacturers Association (1995). National fuel surveys: gasoline and diesel fuel-winter 1995. Detroit:AAMA, 1995. Crump, KS. (I-994). Jtiskofbenzene-induced leukemia: a sensitivity analysis ofthe Pliofllm cohort with additional follow-up and new exposure estimates. J Toxicol Environ Health 42:-219-242. Forni A. (1996). Benzene-induced chromosomal aberrations: A follow-up study. Environ Health Perspect 104: 1309~312: -Gut, I, Nedelcheva V, Soucek, P, Stopka P; and Tichavska B. (1996). Cytochromes_F450 in benzene metabolism and involvement oftheir metabolites and reactive oxygen species in-tcxicity. Environ Health Perspect (104): 1211-1218. Hayes RB, Yin SN, Dosemeci Metal (I-996)._ Benzene and the dose-related incidence ofhematologic neoplaslll-in China. J Natl Cancer Insti 89: 1065-1071. BP-00011623 Rinsky RA, Smith AB, Hornung Ret al. (1987). Benzene and leukemia: an epidemiologic risk assessment. N Engl J Med 316: l044-1050. Rothman N, Li GL, Dosemici Metal. (1996). Hematotoxicity among Chinese workers heavily exposed to benzene. Am J lnd Med 29: 236-246. Smith MT. (1996). The mechanism ofbenzene-induced leukemia: a hypothesis and speculations on the causes of leukemia Environ Health Perspect (104): 1219-1225. Wallace LA (1996). Environmental exposure to benzene: an update. Environ Health Perspect 104: 1129-1136. Witz G, Zhang Z, Goldstein BD. (1996). Reactive ring-opened aldehyde metabolites in benzene hematotoxicity. Environ Health Perspect (104): 1194-1199. Yin SN, Linet MS, Hayes RB, et al. (1994). Cohort study among workers exposed to benzene in China: I. General methods and resources. Am J IndMed 26: 383-400. BP-00011624 Recent Findings and New Initiatives for Epidemiologic Research on Benzene Otto Wong. Sc.D.1.2 1.-ChiefEpidemiologist, Applied Health Sciences, Inc. 181 Second Avenue, Suite 628 San Mateo, California 94401, USA 2. Adjunct Professor ofEpidemiology and Biostatistics Tulane University Medical Center New Orleans, Louisiana, USA Presented at 1998 Benzene State ofthe Science Workshop I University of Ottawa Ottawa, Canada December 17, 1998 Summary of presentation Recent epidemiologic research on benzene consists of (1) updated analyses of workers elq)osed to benzene attwo facilities in Ohio, who were involved in the manufacture ofPliofilm, (2) analyses of workers in the petroleum industry in North America, Australia and Europe, who were exposed to benzene and other petroleum products, and (3) workers from a broad spectrum of industries in China, who were exposed to benzene and a wide variety ofchemicals. Results from the original Pliofilm study were reported by Infante et al. (1978), and -I Rinsky et al. (1981 and 1987). Based on mortality ascertainment through 1981, Rinsky et al. (1987) reported a significant increase in risk as well as an exposure-response relationship between cumulative benzene exposure and leukemia (an cell types combined). An increased risk of multiple myeloma was also reported, but no exposure-response relationship was found. Subsequently, mortality in the Pliofilm cohort was updated through 1987. Based on an analysis on leukemia (all cell types combined), Paxton et al. (1994) concluded that a significant increase in leukemia risk occurred at exposure above 50 ppm-years. However, based on an analysis of acute myeloid leukemia (AML}, Wong ( 1995) concluded that a significant increase in AML occurred at a much higher level (200 ppm-years based on the set of lowest estimates), and that the slope of the exposur-e-response curve was much steeper. No additional death from multiple myeloma was reported in the-update, and the risk was-no longer significant. To examine the-effect of an alternative exposure metric, Schnatter et al. ( 1996a) analyzed the Pliofilm study using the long-term average concentration associated with the maximally exposed job of each worker. Using the lowest exposure estimates, Schnatter-et al. (I996a) concluded that the critical cancentration ofbenzene exposure for an increased risk of AML was.between 20 and 25 ppm. Benzene was the principal- exposure at the Pliofilm-plants, and exposure levels were relatively high. Exposure estimates in_ the Pliofilm study have been subjected to extensive BP-00011625 review, and three different sets of estimates have been developed. Because of the relatively high exposures and the large number of AML cases, the Pliofilm- study will likely continue to provide valuable information on benzene epidemiology, especially for AML. The cohort is now ready for a 10-year update. However, discrepancies in exposure estimates have and will continue to hamper the interpretation. In addition, the small number -of cases of non-AML leukemias limits the statistical power ofthe study. Cohort studies of petroleum workers (primarily refinery and distribution workers) have been updated periodically. These workers were exposed to benzene and benzene-containing products. Unfortunately, few studies of petroleum workers have quantitative benzene exposure estimates, and job categories and duration of employment have often been used as surrogates for exposure. Exceptions were studies based on distribution workers in the US, Canada and the UK. In these studies, estimates of exposure to gasoline (in terms of total hydrocarbons) or benzene were provided (Smith et al., 1993; Wong et al., 1993; Schnatter et al., 1996b; Rushton and Romaniuk, 1997; Wong and Raabe, 1997a; -W-ong et al, 1999). In general, benzene exposure levels in the petmleum industry are relatively low, especially after 1950. For example, based on more than 1,700 samples collected between 1973 and 1982 at a large refinery in Texas, 89% full samples (sampling time > 4 hours, with the majority > 7 hours) were less than 1 ppm, 10% between 1 and 10 ppm, and only a few samples above 10 ppm (Tsai et al., 1983). Overall reviews and meta-analyses by cancer site of petroleum studies have been reported previously (Wong and Raabe, 1989, 1995 and 1997b). For cell-type specific leukemias and other lymphopoietic cancers such as multiple myeloma. because of the small number of deaths in each study and the broad and overlapping categories used in US mortality statistics, separate results were not reported in many original reports of cohort studies in the US. Recently Wong and Raabe {1995 and 1997) reported results for cell-type specific leukemias and multiple myeloma for individual studies as well as for the combined database, which consisted of more than 250,000 workers and covered an observation period of 55 years (1937-1991). No increased mortality from cell-type specific leukemias or multiple myeloma was found. These cohort studies of petroleum workers (particularly those in the US, Canada and Australia) will continue to be updated in the future. For large cohorts, nested case-control studies should be considered. Whenever possible, a thorough exposure assessment should be an integral part of all nested_ case-control studies. Currently, several cohort studies are being updated. In addition, an_overall review and an updated meta-analysis by cancer site of all cohort studies in the petroleum industry are now underway. In 1979-1981 the Institute of Occupational Medicine, Chinese Academy of Preventive Medicine (C.APM), carried out a national survey in China, in which more than 500,000 workers were identiffed to have been exposed to benzene, and a benzene poisoning prevalence of0.51% was reported (Yin et al., 1981__a). The 95% range of benzene concentrations at workplaces were 0.06-844.74 mg/m3 {0.02-266 ppm). Subsequently, 28,460 of these exposed workers from a variety of industries in 12 cities and 28,257 unexposed workers from the same cities were included in a historical cohOit study (the "CAPM' study). The mortality of the cohort was observed between 1972 and 1981, and the results were reported in a number of publications (Ym and Li, 198-6; Yin et al., 1987b; Yin et at., 1989). A total of 25 leukemia deaths _and 5 incidence cases were reported among -the exposed workers, while 4 deaths among the unexposed workers were attributed to leukemia. The standardized mortality ratio-(S'".MR) of leukemia for the exposed workers in comparison to the unexposed workers was 5.74 (p < 0.01). No exposure-response 2 BP-00011626 .- analysis was performed, but the authors reported actual benzene measurements from locations -where leukemia deaths and cases were observed (Ym and Li, 1986; Yin et at., 1987b; Yin et at., 1989). Exposure levels reported were as high as 5,508 mg/m3 or 1,732 ppm. Since- 1987, the US National Cancer Institute (NCI) has coUaborated with CAPM in expanding and updating the Chineseoenzene study. The previous study was expanded to include 74,828 benzene-exposed workers and 35,805 unexposed workers who were employed for any length oftime between 1972 and 1987 at 712 factories in 12 cities (the "CAPM-NCf' study). The general methods and resources ofthe expanded study were described by Ym et at. (1994). In a second paper, an historical benzene exposure assessment to characterize benzene exposure profiles ofthe cohort members was outlined (Dosemeci et al., 1994). Based on comparisons between the exposed and unexposed workers, increased mortality risk ratios (RR) were reported for a variety of cancers: 2.4 for nasopharyngeal cancer, 1.8 for esophageal cancer, 1.2 for cancer ofthe liver and gaD bladder, 1.4 for lung cancer {1.5 in men and 1.0 in women), 1.3 for brain tumors, 4.5 for lymphoma, and 2.3 for leukemia (Yin et al., 1996). Some exposure-response analyses in terms of cumulative exposure (ppm-years) for selected disease categories were reported by Hayes et al. (1996). Both lung cancer and the broad category of hematopoietic malignancies showed a significant upward trend (p = 0.01). For workers with more than 400 ppm-years of benzene exposure, the RR for lung cancer was l.7 and that for hematopoietic malignancies was 2.0. In another paper, Hayes et al. (1997) reported dose-related incidence of hematologic neoplasms. For leukemia, the RR was 2.5 (95% CI: 1.2-S.l). For acute non-lymphocytic leukemia (ANLL), the RR was 3.0 (95% CI: 1.0-8.9). The RR for non-Hodgkin's lymphoma (NHL) was 3.0 (95% CI: 0.9-10.5). The authors reported significant upward trends for ANLL with increasing average exposure, and with increasing constant exposure. The trend between ANLL and cumulative exposure was close to, but did not achieve, statistical significance (p = 0.06). For Nlll.., significant trends were reported for average exposure, duration of exposure and cumulative exposure. The highest Nffi.. risk (RR = 7.8) was observed among "chemical" workers, which included "organic, insecticide, and benzene production workers." Although the Chinese benzene study was one of the largest studies in terms of number of exposed workers, some results were based-on comparisons to small numbers of cases for disease categories ofinterest in the unexposed group. For example, there were only 4 ANLL cases and 3 NH!:, cases among the unexposed workers. For the Chinese benzene study, results depended on not only the number of deaths in the exposed group but also the number of deaths in the unexposed group. The exposed workers were emplQyed in a variety of industries. In the-CAPM study, 64% of the exposed workers were either painters or paint production workers (Yin et at., 1987b). In terms of exposures, only 5% were exposed to benzene only (Yin et al., 1987a). The potential problem of concomitant exposures complicated the inte:-pretation of the results in relation to benzene exposure (for example, the Nffi.. increase in workers exposed to both benzene and insecticides). Comparing to the unexposed workers, the exposed workers were reported to have increased risk in a variety of diseases. For example, in the original CAPM study, benzene-exposed workers experienced a total mortality risk of 2-fold when compared to unexposed workers. Fer all cancers, the mortality risks were 2.5-fold for exposed male workers and 1.8-fold for exposed female workers, when compared to their unexposed counterparts. Even in the expanded CAPM- 3 BP-00011627 ------- NCI study, some occupational groups (such as chemical or rubber manufacturers, painters or paint manufacturers) still exhibited significant increases from total mortality or all cancers (Li et al., 1994). It seemed unlikely that benzene was responsible for all these increases. No other studies have demonstrated such a broad-mortality increase among benzene workers. More specifically, increased mortality risks among exposed male workers-when compared to unexposed workers were reported for many specific cancer sites, which have not been demonstrated to be related to benzene exposure in other studies; including lung cancer, liver cancer, stomach cancer, intestinal cancer, esophageal cancer, and- nasopharyngeal cancer. For example, no study has ever demonstrated an association between benzene and nasopharyngeal cancer. These observations raised potential concerns regarding the comparability of the unexposed workers, potential under-ascertainment of cancer among the unexposed workers, and confounding exposures (or other risk factors) in the exposed group (Yin et al., 1989). Cigarette smoking-has been found to be a risk factor for leukemia (particularly AML) in a number of epidemiologic studies. The fact that lung cancer risk was significantly elevated (in men) and that a significant exposure-response relationship in terms of cumulative exposure to benzene was observed among the exposed workers in the Chinese benzene cohort caJied for an examination ofthe potential confounding by smoking (Yin et al., 1989 and 1994). Based on case-control studies ofleukemia in Shanghai, Lin et al. (1987, 1991) identified a -number of significant risk fact-ors: vinyl chloride (odds ratio, OR = 11.60), organic phosphate (OR = 2.15), toxic heavy metals (.OR = 3.90), and radiation therapy or occupational exposure to radiation (OR= 6.58). The risk associated with the use of chloromycetin (chloramphenical) was also elevated (OR= 1.69, p = 0.06). In a large series of leukemia cases in the Henan Province, China, a large percentage of patients had a history of using chloromycetin (Wang et al., 1984). In the same series, the use of other medications was also reported. These potential confounding exposures or risk factors complicated the interpretation of findings (Yin et al., 1989 and 1994). One of the areas of uncertainty in the Chinese benzene study involved the exposure estimates. Comparing the assumptions used in the development of estimates and the exposure estimates themselves to actual data reported -identified a number of inconsistencies and/or inadequacy of exposure. categories. For example, the actual average exposure oi spray painters (full-day exposure) at a factory in Shanghai in 1965-1969 was 331 mgim3 (104 ppm), whereas the estimate developed by Dosemeci et al. (199.4) for spray painters in the same time period was 22.1 ppm. There are other similar examples. It appeared that the exposure estimates were consistently lower than the actual exposure data. The "indirect validation" conducted by Dosemeci et al (1996), by definition, could not validate the absolute values of the estimates. Dosemeci et al. (1994) recognized that little confidence could be attached to the estimates: only 2% of the estimates for the time intervall949-1959 and only 6% of the estimates prior to 1975 were rated in the "higJ\ confidence., category. Exposure-response analyses based on these-exposure estimat-es should be interpreted with extreme caution. Both CAPM and NCI have made substantial efforts in studying the relationship between benzene exposure and malignancies. The CAPM-NCI study is certainly one of the largest occupational studies:- Because of the large number of- workers who were exposed to relatively high levels ofbenzene (compared to, for example, levels at refineries in the US), data from-China can pr-ovide further understanding of the health effects of benzene. Recent investigations of biomarkers will likely provide new insights as well (Rothman et al., 1996). However, for the results to be more reliable, the issues raised above regarding the data need to be addressed. 4 BP-00011628 Based on a review of the current benzene literature, considerations should be given to the following areas in future benzene epidemiologic research: exposure range in relation to study objective, exposure- estimates for individual workers, adequacy ofexposure categories, concomitant exposures to other chemicals, non-occupational risk factors-, specific leukemia cell-types, other hematologic malignancies in addition to leukemia, and incorporating biomarkers or biomonitoring data in epidemiologic studies. References Dosemeci M, Li GL, Hayes RB, et aJ. (1994): Am.JlndMed26:401-411. Dosemeci M, Yin SN, Linet M, et al. (1996): EllViron Health Persp 104(Suppl6):1343-1347. Hayes RB, Ym SN, Dosemeci M, et al. (1996): Environ/ Health Persp 104(Suppl6):1349-1352. Hayes RB, Ym SN, Dosemeci M, et aJ. (1997): JNC! 89:1065-1071. Infante PF, Rinsky RA, Wagoner JK, et al. {1977): Lancet 2:76-78. Li GL, Linet MS, Hayes RB, et al. (1994): J Occup Med36:875881. Lin GW, Lin PT, Ting Z, et al. (1987): Chinese Joumal ofHematology 8:713-715. [in Chinese] Lin GW, Lin PT, Wang C, eta!. (1991): Shanghai Medical Joumal 14:497-500. [in Chinese] Paxton MB, Chinchill VM, BretrSM, et al. (r994): Risk Analysis 14;.147-154. RinskyRA, YoungRJ, SmithAB, etal. (1981):AmJ/ndMed2:211-245. RinskyRA. SmithAB,HomungR, etal. (1987):NewEnglJMed316: 1044-1050. Rothman N, Smith MT, Hayes RB, et al. (1996): Environ Health Persp 104(Suppl6):1365-1370. Rushton L, RomaniukH (1997): OccupEnvironMed54:152-156. Schnatter AR, Nicolich-MJ, Bird MG (1996a): Risk Analysis 16:833-840. Schnatter AR, Armstrong TW, Nicolich MJ, eta!. (1996b): Occup Environ Med 53:773-781. Smith-TJ, Hammond SK, HallockM, et al. (1993): Environ Health Persp lOl{Suppl. 6):13-21. Tsai SP, Wen CP, Weiss NS, et al. (1983): JOccupMed25:685-692. Wang HP, et al. (1984): Chinese Journal ofHematology 5:217-228. [in Chinese] Wong 0;-Raabe GK (1989): Am J Ind Med 15:28-3-310. Wong 0, Harris F, Smith TJ (1993): E11Viron Health Persp IOI(Suppl. 6t63-76. Wong 0 (1995): Occup-EnvironMed 52:380-384. Wong 0, Raabe GK (1995): Reg Toxicol Pharmaeol21:301-32L Wong 0, Raabe GK (l997a): Occup Environ Med 55:360-362. Wong 0, Raabe GK (1997b): Reg Toxicol Pharmacol26:188-199. Wong 0, Trent L, Harris F (1999): Occup Environ Med 56 [in press]. Yfn SN, Li GL (l98o): Chinese J Ind Hyg_Occup Diseases 4{4):204-207. [in Chinese] Yin SN, Li Q, Liu Y, et al. (1987a): Brit J Ind Med 44:192-195. Ym SN,Li GL, Tain FD, et al. {1"987b): Brit J lndMed 44:124-128. Yin SN, Li GL, Tain FD, et al. (1989): Environ Health Persp 82:207-213. Yin SN, Linet-MS, Hayes RB, et al. (1994): Am J lndMed26:383-400. Yin SN, Hayes RB, Linet MS, et al. (1996): Am J lndMed29:227-235. s BP-00011629 ISSUES FOR DISCUSSION: BENZENE INDUCED LEUKEMIA-HUMAN STUDIES David Bayliss and Babasabeb Sonawane National Center for Environmental-Assessment U.S. Environmental Protection Agency Washington, D.C. 20460 National Cancer Institute/Chinese Epidemiologic Study With respect to the Chinese cohort (Dosemeci et al., 1994; Hayes et aJ., 1996, 1997; Yin et al., 1987, 1989, 1994, 1996), the following issues are of concern: 1) What ~~were the socioeconomic conditions prevalent in the members of the cohort i.e. factory workers and how are they different from those of benzene workers in the United -states? Were there environmental factors i.e. diet, lifestyle habits, etc. of the Chinese cohort that were different from those ofU:S. benzene workers studies (e.g. Rinsky-et aJ., 1981, 1987) that could have a differentially adverse impact upon health conditions in these workers. How were they controlled for in the analysis? What were the known or potential carcinogens other than benzene in the workplace of the Chinese that could have produced confounding effects? .2) What was the evaluation and decision- process used in each factory to assign estimated exposures to individua1 factery workers where no actual measurements were available? In the Dosemeci et-al., 1994 Study, it is stated that 97% of the early estimates were not based upon actual measurements. What safeguards were employed to insure that the assigned exposures at the factory level were not biased but reflect the best judgement of the experts concerning potential exposures that existed at the time of the employment of the worker in each factory? 3) Othe!Conditions, for which the risks were- elevated, i.e. non-Hodgkin's lymphoma, myelodysplastic syndromes and hematologic neoplasms need to be conftnned in other-studies. The latter two appear to be grouped categories containing an assortment of blood related disorders including cancer. Can the risk of individual unique conditions be determined? BP-00011630 Biomarkers ofLeukemia Risk Our main concern here is whether biological responses reported as biomarkers are really indicators of a population at a higher risk of leukemia (biomarker<>..-of effect) or are they just indicative of a physiologicaJ response that may not be associated with a higher risk ofcancer but perhaps only markers of exposure. Insights from Petroleum Studies With respect to petroleum worker studies, we have noted several observations. Most of these involve very low exposures to benzene, generally uneer I ppm. Most of them show little or no excess risk of leukemia, even ANLL. And, of course, there is little data addressing exposures between 1 ppm and 20 to 40 ppm (cumulative), a range that remains largely without information concerning sensitivity of risks (except perhaps the newer Chinese factory workers cohort). In the largest -amalgam of petroleum -workers studied thus far, Raabe and Wong (L996) studied a combined cohort of 208,000 workers (19 plants). The reported average exposure to benzene was-found to be only 0.22 ppm.-in such workers according to Runion (1988) based upon nearly 15,000 samples. The average cumulative exposure received if a worker remained employed in this industry for 40 years would be .22 ppm X 40 years or 8.8 ppm-years. More than likely, very few of the workers ever achieved a cumulative exposure of 200 ppm-~ears in this iildustry-wide study. The comparison background population that provided expected deaths in this study may have also sustained exposures that hovered around or close to this level of 0.22 ppm found in the "exposed" cohort. If this is so, then one wculd- not expect to detect an increase in the risk of lung cancer in this study. As a matter of fact, the observed cases were not significantly different from expected.. An interesting theory was raised by Dr. Schnatter's 1996 study_ of petroleum -workers (Schnatter et al., 1996}, and-that is, that peak exposur-es (excursions) may be more closely associated with the development of leukemia, rather than long term cumulative exposures. Dr. Schnatter used the median of the sets of exposure estimates-of Rinsky, Grump ancFPaustenback to develop a new set of indices of exposure per person. An "average" total concentrat-ion per person was determined from the job category with BP-00011631 the greatest exposure (maximum) and of longest duration. This method made it possible to isolate subgroups with less exposure to specified concentrations of benzene and examine the relative risks to each ofthese subgroups. The ioea was that these subgroups were unlikely to be exposed to concentrations greater than a specified concentration. Excursions and peak exposures could be potentially reduced in such groups and a better indication of what level of cumulative exposure is associated with an increased risk of leukemia could be ascertained. This assumes that it is the peak exposures that are responsible for the risk and eliminating them from the analysis would reduce any potential skewing of the results by the presence of peak exposures in the data. One difficulty with this idea is that the pliofilm cohort is not powerful enough at low doses to detect an elevated risk even ifone were there. One possible data source that might be considered in the future for this type of analysis is the Chinese cohort data. Utilizing this data set might provide a1arge enough cohort at the lowest exposure levels that epidemiologists would no longer be plagued by small population sizes and consequently a lack of sensitivity at low exposures. New Initiatives for Epidemiologic Research An issue for discussion should be how a new epidemiology study would reduce uncertainties that remain in.current epidemiology studies and by reducing them would help to improve the cancer risk assessment at low doses. Again, the cohort must be large,_in order to detect significant risks at low doses of exposure to benzene. Secondly, it must exhibit a wide range of exposures to benzene, especially between I ppm .and 50 ppm. and thirdly, it must be free of other confounding exposures as much as possible that could potentially influence the risk. The problems with exposure information are the biggest concern for risk assessors and incorporation of biochemical! molecular biomarkers of exposure/ effects in mode of action. The estimated unit risk estimate from the Rinsky et al., 1987 study could change by as much as I or 2 orders of magnitude depending on whieh set of estimated exposures are used to derive the unit risk based on his data alone. If there were some method for deriv-ing fairly-reliable exposure estimates it would reduce the uncertainty ofthis parameter considerably. - - - - ---. ---------- BP-00011632 References Dosemeci, M; Li. GL; Hayes. RB; et al. (1994) Cohort study among workers exposed to benzene in China: ll. Exposure assessment. Am J lnd Med 26:401-411. Hayes, RB; Yin, SN; Dosemeci., MS; et al (1996) Mortality among benzene-exposed workers in China. Environ Health Perspect 104(supp16): 1349-1352. Hayes, RB; Yin, SN; Dosemeci, MS; et al. t1997) Benzene and the dose-related incidence ofhematologic neoplasms in China. J Nat Cancer lnst 89:1065-1071. Raabe and Wong (1996) Leukemia Mortality by Cell Type in Petroleum Workers with Potential Exposure to Benzene. Environ Health Perspect 104(suppl 6): 1381-1392. Rinsky, RA; Young, RJ; Smith, AB. (1981) Leukemia in benzene workers. Am J lnd Med 2:217-245. Rinsky,- RA; Smith, AB; Hornung, R; et al. (1987) Benzene and leukemia: an epidemiologic risk assessment N Engl J Med 316:1044-1050. Runion, HE. (1988) Occupational exposures to potentially hazardous agents in- the petroleum industry. Occup Med: State ofthe Art Reviews 3:431-444. Scbnatter, AR; Nicelich, MJ; Bird, MG. (1996) Determination of leukemogenic benzene exposure concentrations: refined analyses of the Pliofilm-cohort. Risk Anall6:833-840. Yin, SN; Li, GL; Tain, FD; eLal. (1987) Leukaemia in benzene workers: A retrospective cohort study. Br J Ind Med 44:124-128. BP-00011633 Yin. SN; Li, GL; Tain, FD et al. (1989) A retrospective cohort study of leukemia and other cancers in benzene workers. Environ Health Perspect 82:207-213. Yin. SN; Linet, MS; Hayes, RB; et al. (1994) Cohort study of cancer among benzeneexposed workers in China: I. General methods and resources, Amer J Ind Med 26:383400. Yin. SN; Hayes, RB; Linet, MS~ et al. (1996) A cohort study of cancer among benzeneexposed workers in China: Overall results. Am J Ind Med 29:227-235. (The views expressed are that of the authors and do not necessarily represent EPA policy or endorsements) BP-00011634 1998 BENZENE STATE OFTHE-SCIENCEWORKSHOP University of Ottawa.lntitute1e:--Population Health DELEGATE LIST Alatia, Raghda Dept. of Epidemiology University of Ottawa 451, Smyth Rd. Ottawa ON K1H 8M5 Tel: 613-562-5800 x 8292 Fax: 613-562-5465 E-Mail: ralatia@zeus.med.uottawa.ca Andersen, Melvin ICF Kaiser, K.S. Crump-Group, P.O. Box 14348 Research Triangle Park, NC 27709 Tel: 919-547-1723 Fax: 919-547-1-710 E-Mail: andersenme@aol.com Atkins, -1--tarold Ottawa General Hospital Division of Hematology 501 Smyth Rd., rm 7209 Ottawa ON K1H 8L6 Tel: _613-737-8803 or 8152 Fax: 613-737-8861 E-Mail: Band, Pierre Health Canada 1001 St. Laurent West Longueuil-, Quebec J4K 1C7 Tel: 450-64o-1353 ext 306 Fax: 450-928-4102 E-Mail: pierre::.band@hc-sc.gc.ca Bayliss,. David U.S. Environmental Protection Agency 401 M Street SW #86230 Washington DC 20460 Tel: 202-564-3294 Fax: 202-565-0078 E-Mail: no e-mail BP-00011635 Beatty, Patrick Chevron, Research and Technology Co. 100, Chevron-way Richmond CA 94802 Tel: 510-242-7037 Fax: 510-242-7022 E-Mail: pwbe@chevron.com Bird, Michael Exxon Biomedk:al Sciences Inc. CN 2350, Mettlers Road East Millstone NJ 08875~350 Tel: 73~-873-6067 Fax: 732-873-6009 E-Mail: mgbird@fpe.erenj.com Bond, Gary Phillips Petroleum Company Risk Assessment and Toxicology 12 A 2 Phillips Building Bartlesville OK 74004 Tel: 918-661-0597 Fax: 918-662-1139 E-Mail: gpbond@ppco.com Burnett, Don Amoco Corp 28100 Torch Parkway Suite 400 Warrenville IL 60555-4015 Tel: 630-836-5666 Fax: 630-836-5717 E-Mail: drriburnett@amoco.com Chen, Chao U.S. Environmental Protection Agency 401 M. St S.W. Washington DC 20460 Tel: 202-564-3277 Fax: 202-565-0079 E-Mail: eben .chao@epamaH:-gov I BP-00011636 Cole, Maxine Department-of Epidemiology and Medicine University of Ottawa 451, Smyth Ottawa ON K1H 8L6 Tel: 613-562-5800 Fax: 613-562-6455 E-Mail: cole@labelc.med.uottawa.ca Cox, Anthony Cox Associates 503 Franklin St. Denver CO 80218 Tel: 303-388-1778 Fax: 303-388-0609 E-Mail: tony@cox-associates.com Divine, Barbara Texaco Inc. Texaco Heritage Plaza P.O. Box 1404 Houston TX 77251 Tel: 713-752-6290 Fax: 713-752-4652 E-Mail: divinbj@texaco.com Eastmond, David University of California- Riverside- Environ. Tox. Program 5419 Boyce Hall Riverside CA 92521 Tel: 909-787-4497 Fax: 909-787-3087 E-Mail: david.eastmond@ucr.edu Erdal, Serap EOSHI Rutgers University 170 Frelinghuysen Rd. Piscataway NJ 088542 Tel: 732-235-3294 Fax: 732-235-4569 E-Mail: erdal@eoshi.rutgers.edu BP-00011637 Evans, Marina USEPA PRBIMD74 Research Triangle Park NC 27711 Tel: 919-541-0838 Fax: E-Mail: 919-541-5394 evans.marina@epamail.epa.gov French, John NIEHS P.O. Box 12233, MD F105 Research Triangle Park NC 27709 Tel: 919-541-2569 Fax: 919-541-1460 E-Mail: french@niehs.nih.com Frisch, Jonathan API 1220 L. Street N.W. Washington D.C. 20005 Tel: 202-682:.8480 Fax: 202-682-8270 E-Mail: frischj@api.org Goddard, Michael Health Canada AIL088B1 Tunney's Pasture Ottawa ON K1AOL2 Tel: 613-954-0169 Fax: 613-941-8632 E-Mail: mgoddard@ehd. hwc.ca Goldstein, Bernard UMDNJ EOHSI 170 Frelinghuysen Rd. Piscataway NJ 08854 Tel: 732-445-0205 Fax: 732~-0131 E-Maif: bgold@eohshrutgers.edu BP-00011638 GranviUe, Geoffrey Shell Canada Ltd. 400, 4th Ave. S.W. Calgary AB T2P 2H5 Tel: 403-691-2220 Fax: 403-691-2224 E-Mail: granvil@shell.ca Hayes, Richard U.S. National Cancer Institute 9000 Rockville Pike, EPN 418 Bethesda MD 20892 Tel: 301-435-3973 Fax: 301-402-1819 E-Mail: hayesr@epadce.nic.nih.gov Healy, Laura CUT 6 Davis Rd. Research Triangle Park NC 27709 Tel: 919-~8-1380 Fax: 919-558-1300 E-Mail~ healy@ciit.org Heaney, Mark MemoriatSJoan-Kattering-Cancer- Center 1275, York Ave. NewYork NY 10021 Tel: 212-639-2275 or 7871 I Fax: E-Mail: 212-772-8550 m-heaney@ski.mskcc.org Henderson, Rogene Lovelace Respitory Research Institute P~O.Box 5890 Aiberqurque NM 87185 Tel: 505-845-1164 Fax: 505-845-1198 E-Mail: rhenders@lrri.org BP-00011639 Henry, Carol API 1220 L Street NW Washington DC 20005 Tel: 202-682-8308 Fax: 202-682-8270 E-Mail: henrycj@api.org Hoffman, Matffiew Dept. of Toxicology Rutgers University 176 Frelinghuysen Rd. Piscataway NJ 08854-8020 Tel: 732-445-3752 Fax: 732-445-0119 E-Mail: matthoff@eden. rutgers. edu Hughes, Kathy Health Canada Environmental Health Centre Tunney's Pasture, PL 08028_1 Ottawa ON K1AOL2 Tel: 613-957-1250 Fax: 613-954-2486 E-Mail: kathy_hughes@hc-sc.gc.ca Irons, Richard University of Colorado -Health Sciences Center 4200 E. 9th Ave C238 Denver CO 80262 Tel: 303=315-7170 Fax: 303-315-7223 E-Mail: richard.irons@uchsc.edu Kacew, Sam Faculty of Medicine University of Ottawa 451, Smyth Ottawa ON K1H 8M5 Tel: 613-562-5800 x 8357 Fax: 613-562-5434 E-Mail: skacew@uottawa.ca BP-00011640 Kalf, George Thomas Jefferson University- Dept. Biochemistry and Pharmacology 102.0 Locust St., Jefferson Alumni Hall Philadelphia PA 19006 Tel: 215-503-0203 Fax: 215-503-2365 E-Mail: george.kalf@mail.tju.edu Keating, Armand Ontario Cancer Institute Princess Margaret Hospital Dept. of Medical Oncology and Hematology 6tO, University, suite 5-211 Toronto ON M5G2M9 Tel: 416-340-3993 Fax: 416-340-5087 E-Mail: akeating@torhosp.toronto.on.ca Keefe,_Roger Imperial Oil Ltd. 111 St. Clair Ave. West Toronto ON M5W-1K3 Tel: 416-968-4435 Fax: 416-968-8415 E-mail: rtkeefe@netcom .ca Kester, Janet Dames & Moore 7254 W. Piute Dr. I -sedalia, 80135 CO Tel: 303-688-6497 Fax: 303-688-1135 E-Mail:denjek@dames.com Kreiger, Gary R. University of Colorado School of Pharmacy 633, 17th St., suite 2500 Denver CO 80202-3625 Tel: 303-299-7848 Fax: 303-299-7901 E-Mail: dengrk@dames.com BP-00011641 Krewski, Dan University of Ottawa. Department of Epidemiology 451 Smyth Rd Ottawa ON K1H 8M5 Tel: 613-562-5800 ext 8261 Fax: 613-562-5465 E-mail: dkrewski@uottawa.ca Krishnan, Kannan University of Montreal Medecine du Travail et Hygiene du Milieu CP 6128, Succursale, Centre-Ville Montreal Quebec H3C 3J7 Tel~ 514-343-6581 Fax: 514-343-2200 E-Mail: krishnak@ere.umontreal.ca Kroese, E. Dinant . RIVM Laboratory of Health Effects Researcll National Institute of Public Health & the Environment Antoine van Leeuw.enhoeklaan 9, P.O. Box 1 3720 BA Bilthoven NETHERlANDS Tel: 31-30-274-3483 Fax: 31-30-214-4446 E-Mail: ED.Kroese@rivm.nl Larson, Richard University of Chicago 5841 So. Maryland Ave., MC 2115 Chicago IL 60637 Tel: 773-702-6783 Fax: 773-702-0963 E-Mail: ralarson@mcis. bsd. uchicago.edu Laskin, Debbie Rutgers University EO.HSI Toxicology Division 170 Fre!inghuysen Rd. Piscataway NJ 08854-8020 Tel: 732-445-5862 lab 4702 Fax: 732-445-2534 E-Mail: laskin@eoshi.rutgers.edu BP-00011642 Linet, Martha S., M.D. National Cancer Institute Dvision of Cancer Epidemiology & Genetics 6130 Executive Blvd. EPN room 408 Bethesda, Maryland 20892-7362 Tel: 301-496-6600 Fax: 301-402-0207 E-Mail: linetm@epndce.nci.nih.aov Luebeck, Geor Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N. P.O. Box 19024 Seattle WA 98109-1024 Tel: 206-667-4282 Fax: 206-667-7004 E-Mail: j@trex.fhcrc.org Mackerer, Carl Mobil Business Resources Corp. P.O. Box 310 Paulsboro NJ 08066 Tel: 609-224-4630 Fax: 609-224-4652 E-Mail: carl_r_ mackerer@e.maiLmobil.com -McClellan, Roger CIIT P.O. Box 12137 6 Davies -Dr. Research X.riangle Park NC 27709 Tel: 919-558-1-2-02 Fax: 919-558-1400 E-Mail: mcclellan@ciit.org Meek, Betty Health Canada Environmental Health Centre- Tunney's Pasture, rm 217 Ottawa ON K1A OL2 Tel: 613-957-3129 Fax: 6'13"-954-2486 E-Mail: bette_meek@hc-sc.gc.ca BP-00011643 Murray, Neil Imperial Oil, Ltd. 111 St. Clair Ave., West Toronto Ontario M5W1K3 Tel: 416-966-4320 Fax: 416-968-8415 E-Mail: neil.murray@iol.ca.sprint.com Oliver, David Shell Oil Company 711, Louisiana Houston TX 77002 Tel: 713-220-8114 Fax: 713-236-0822 E-Mail: doliver@aking up.com Portier, Chris National Institute Environmental Health Science 111 TW Alexander Dr. Bid 101 South Campus RMA-344 Research Triangle Park NC 27709 Tel: 919-541-4999 Fax: 919-541-1479 E-Mai: portier@niehs.nig.gav Raabe, Gerhard Mobil-Global Medical Services 2215, Aquetong Rd. New Hope PA 18938 Tel: 215-8>2-5718 Fax: 215-862-3551 Sheryl Fax: 703-846-7424 E-Mail: gerhard_k_raabe@email.mobil.com Raza, Azra Rush Presbyterian-St Lukes Medical Center Cancer Institute 2242 W. Harrison St. Suite 108 Chicago IL 60612 Tel: 312-455-8474 Fax: 312-455-8479 E-Mail: araza@rush.edu BP-00011644 Recio, Leslie CIIT 6 Davis Rd. Research Triangle Park NC I 27709 Tel: 919-558-1329 Fax: 919-558-1300 E-Mail: recio@@ciit.org Richmond, Martha Health Effects Institute 955 Massachusetts Ave. Cambridge MA 02139 Tel: 617-876-6700 Fax: 617-876-6709 E-Mail: mrichmm@healtheffects.org Rinsky, Robert NIOSH, CDC 4676 Columbia Parkway, r-44 Cincinnati OH 45226 Tel: 513-841-4131 Fax: 513-841-4470 E-Mail: rar1 @cdc.gov Rodan, Bruce Senior Health Scientist Natfonal Center for Risk Assessment 401, M. Street West Washington, DC 20460 Tel: 202-546-3329 Fax: 202-565-0066 Ross, David University of Colorado Health Sciences Center School of PharmacyBox C 238 4200 E. 9ti=J-Ave Denver CO 80262 Tel: 303-315-6077- Fax: -303-315-0274 E-Mail: david.ross@uchsc.edu BP-00011645 Schlosser, Paul GilT P.O. Box 12137 6 Davis Drive Research Triangle Park NC 27709 ::rei: 919-558-1243 Fax: 919-558-1300 E-Mail: schlosser@ciit.org Schnatter, Rob Exxon Biomedical Sciences Inc. Mettlers Road, CN 2350 East Millstone NJ 08875-2350 Tel: 732-873-6016 or 6930 Fax: 732-873-6009 E-Mail: arsch na@fpe.erenj .com Schonwalder, Christopher National Institute of Environmental Health Science P.O. Box 12233 111 Alexander Dr. Research iriangle Park NC 27709 Tel: 919-541-4794 Fax: 919-541-2260 E-Mail: schonwalder@niehs.nih.gov Siemiatycki, Jack University of Quebec lnstitut Armand-Frappier 531 Boulevard des Prairies Laval-des-Rapides, Quebec HH7V 187 Tel: 450-687-5010 Fax: 450-686-5510 E-Mail: Smith, Martyn University of California School of Public Health 140 Warren Hall Berkeley CA 947-20 Tel: 510-642-8770 Fax: 510-642-0427 E-Mail: martyns@uctink4.berkeley.edu BP-00011646 -Snyder, Robert EOSHI Rutgers University Dept. of Pharmacology & Toxicology 170 Frelinghuysen Rd Piscataway NJ 08854-802G- Tel: 732-445-3720 Fax: 732-445-0119 E-Mail: rsnyder@eohsi.rutgers.edu Sonawane,Babasaheb USEPA 401 M. St. SW Washington DC 20460 Tel: 202-564-3292 Fax: 2{)2-565-0078 E-Mail: sonawane.bob@epamail.epa.gov Stern, Bonnie API 1220 L St. NW Washington DC 20005 Tel: 202-682-8000 Fax: 202-682-8270 E-Mail: no e-mail Steup, Dave- EQUIVA Services 12700 Northborough Dr. NAX 130T Houston TX 77067 Tel: 281-874-4966 Fax: 281-775-5022 E-Mail: drsteup@shellus.com Strother, Dale BP Chemicals Inc. 4440 Warrensville Center Rd. Cleveland OH 44-128 Tel: 216-Ss-6-8262 Fax: 216-586-8314 E-Mail: strothde@bp.com BP-00011647 Sunshine, Geoffrey Health Effects Institute 955 Massachussetts Ave. Cambridge MA 02139 Tel: 617-876-6700 Fax: 617-876-6709 E-Mail: gsunshine@healtheffects.org Trush, Michael Johns Hopkins School of Hygiene & Public Health 615 N. Wolfe St. Baltimore MD 21205 Tel: 41 0-955-2973 Fax: 41 0-955-0116 E-Mail: mtrush@jhsph.edu Tushingham, Mark Environment-Canada 1Oth floor, 351, blvd. St-Joseph Hult PQ K1A OH3 Tel: 819-994-051 0 Fax: 819-953-8903 E-Mail: mark.tushing ham@ec.gc.ca Van der Jagt, Richard Associate Professor Ottawa Hospital, General Campus Division of Hematology 7ttt floor, room 7208 -so1, Smyth Rd. Tel: 613-737-8804 Fax: 613--737-8861 E-Mail rvandeoagt@ogh.on.ca Whysrrer, John American Health Foundation 1 DanaRd Va-lhalla NY 10595 Tel: 914-789-7137 Fax: 914-592-6317 E-Mail: whysner@aol.com BP-00011648 WQQg, Otto Applied Health Science 181 2nd Ave. Suite 628 San Mateo CA 94401 Tel: 650-347-7898 Fax: 650-344-6887 E-Mail: ottowong@aol.com Worton, Ronald Ottawa General Hospital Research Institute Ottawa General Hospital Campus 501, Smyth Rd. Ottawa ON K1H 8L6 Tel: 613-737-8802 Fax: 613-737-8803 E-Mail: rworton@ogh.on.ca Zeise, Lauren Office of Environmental Health-Hazard-Assessment 1515 Clay St. Oakland CA 94612 Tel: 510-622-3195 or 3190 Fax: 510-622-3196 E-Mail: lzeise@hw1.cahwnet.gov -I l BP-00011649