Leukemogenesis In Genetically-altered Mouse Models

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Public health concern over ionizing radiation and benzene, a radiomimetic, has increased because of their potential to induce hematopoietic cancers, especially in the very young. Early exposure to radiation and benzene may be associated with a depression of blood forming elements leading to anaplastic anemia, followed by lympho- or myelodysplastic phenotypes that ultimately may lead to leukemia or lymphoma. Multiple routes of exposure (Inhalation, oral and/ or dermal) to benzene may results in a slower rate of delivery and a greater internal dose to susceptible target tissues. At equivalent inhalation and/or oral dose (mg/kg/body weight), more benzene may be expired unmetabolized after administration by the oral route (60%) than by inhalation (14%). Most critical to selection of dose for experimental studies in animal models is the determination of the exposure level that becomes saturating to pathways of detoxification and alters the dose delivered to the hematopoietic stem cell compartment (e.g. bone marrow). For inhalation exposure to mice this is approximately 200 ppm (6 h TWA). Between 5 and 50 ppm benzene (6 h TWA, there is no significant difference between urinary metabolites. Available data also suggests that the ratio of hydroquinone or muconic acid to phenol ratio after inhalation exposure to 50 ppm (6 h TWA) is closer between mice and humans than either rats or Cynomolgus monkeys. This is critical because these may be the most toxic benzene intermediates. Thus, an exposure model benzene induced hematopoietic disease must take into account saturation of benzene metabolism pathways, available data on exposures and high-affinity, low capacity pathways of metabolism that result in the most hematotoxic intermediates. We have been able to show that B6.129-Trp53 haploinsufficient mice exposed to intermittent low levels (100 ppm, 6 h TWA) develop thymic lymphomas rapidly, whereas mice exposed to more frequent and higher levels of benzene(200 ppm, 6 h TWA) develop tumors less rapidly and at a significantly decreased incidence. In these studies, benzene induced lymphomas in the p53 deficient mice were: 1) clonal (T-cell receptor rearrangements were common), 2) showed a pattern of loss or deletions in chromosome 11 carrying the p53 wildtype allele different from sporadic lymphomas, and 3) showed a pattern of dysregulation of critical genes in both the p53 and Rb pathways that affected cell cycle control and population growth and apoptosis. Using hematopoietic stem cell cultures in vitro from B6.129-Trp53 haploinsufficient mice that are are homozygous null for the Cyp2E1 gene (critical to activation of benzene to benzene oxide), we have been able to show an absence of toxicity and reduced DNA damage to pluripotential hematopoietic stem cells compared to mice with wildtype Cyp2E1 genes. Using polyclonal rabbit anti-S-phenylcysteine we are developing assays to determine the level of benzene adduction to the bone marrow compartment to determine the relationship between dose and the dose delivered to the target tissue. This will aid in testing the hypothesis that lower doses of benzene metabolized by the stromal cells of the bone marrow and the toxic metabolites produced locally may be directly acting on the hematopoietic stem cell compartment and initiating tumorigenesis.