Leukemogenesis In Genetically-altered Mouse Models

公众对电离辐射和苯的健康担忧有所增加，因为它们有可能诱发造血癌，特别是在非常年轻的人群中。早期接触辐射和苯可能与造血元素抑制有关，导致再生障碍性贫血，随后是淋巴或骨髓增生异常表型，最终可能导致白血病或淋巴瘤。接触苯的多种途径(吸入、口服和/或经皮)可能会导致较慢的递送速度和较大的易感目标组织的内部剂量。在等量吸入和/或口服剂量(mg/kg/体重)下，口服给药后过期未代谢的苯(60%)可能比吸入(14%)更多。在动物模型中选择实验研究剂量最关键的是确定使解毒途径饱和并改变向造血干细胞室(例如骨髓)输送的剂量的暴露水平。对于小鼠的吸入暴露，这大约是200ppm(6小时TWA)。在5ppm和50ppm苯(6h TWA)之间，尿代谢物之间没有显著差异。现有数据还表明，吸入50ppm(6h TWA)后对苯二酚或肉豆蔻酸/苯酚的比率比大鼠或食蟹猴更接近于人和小鼠。这一点至关重要，因为这些可能是毒性最大的苯中间体。因此，苯致造血疾病的暴露模型必须考虑苯代谢途径的饱和、暴露的现有数据以及导致最大血毒性中间体的高亲和力、低容量的代谢途径。我们已经能够证明，暴露于间歇性低水平(100ppm，6h TWA)的B6.129-Trp53单倍体不足的小鼠迅速发生胸腺淋巴瘤，而暴露于更频繁和更高水平的苯(200ppm，6h TWA)的小鼠发生肿瘤的速度较慢，发病率显著降低。在这些研究中，P53基因缺陷小鼠的苯致淋巴瘤有：1)克隆性(常见的T细胞受体重排)，2)11号染色体上携带P53野生型等位基因的丢失或缺失模式，不同于散发性淋巴瘤，3)P53和Rb通路中关键基因的异常调节模式，影响细胞周期控制和种群生长和凋亡。使用B6.129-TrP53单倍体缺陷小鼠的体外造血干细胞培养，这些小鼠是CYP2E1基因纯合零(对苯氧化活化至关重要)，我们已经能够证明，与野生型CYP2E1基因小鼠相比，没有毒性，并减少了对多潜能造血干细胞的DNA损伤。利用兔抗S-苯半胱氨酸的多克隆抗体，我们正在开发测定苯在骨髓室的加合水平的方法，以确定剂量与给药到靶组织的剂量的关系。这将有助于检验一种假设，即由骨髓基质细胞代谢的较低剂量的苯和当地产生的有毒代谢物可能直接作用于造血干细胞室，并启动肿瘤形成。

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.