Leukemogenesis In Genetically-altered Mouse Models

公众对电离辐射和苯（一种拟放射线）的关注日益增加，因为它们有可能诱发造血癌症，特别是在非常年轻的儿童中。早期暴露于辐射和苯可能与造血因子抑制相关，导致间变性贫血，随后出现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/或皮肤接触）可能导致释放速度较慢，对易感靶组织的内部剂量较大。在同等吸入和/或口服剂量（mg/kg/体重）下，口服给药后未代谢的苯过期（60%）可能多于吸入（14%）。在动物模型中进行实验研究时，选择剂量最关键的是确定使解毒途径饱和并改变输送到造血干细胞室（如骨髓）的剂量的暴露水平。对于吸入暴露于小鼠，这大约是200 ppm（6小时TWA）。在5和50 ppm苯（6 h 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（6小时TWA）暴露后，小鼠和人类之间对苯二酚或鼻屎酸与苯酚的比率比大鼠或食蟹猴更接近。这是至关重要的，因为这些可能是最有毒的苯中间体。因此，苯诱导的造血疾病暴露模型必须考虑苯代谢途径的饱和、现有暴露数据和导致大多数血液毒性中间体的高亲和力、低容量代谢途径。我们已经能够证明，B6.129-Trp53单倍体不足的小鼠暴露于间歇性低水平（100 ppm， 6小时TWA）会迅速发展为胸腺淋巴瘤，而暴露于更频繁和更高水平的苯（200 ppm， 6小时TWA）的小鼠发展为肿瘤的速度较慢，发病率显著降低。在这些研究中，苯诱导的p53缺陷小鼠淋巴瘤表现为：1)克隆（t细胞受体重排常见），2)携带p53野生型等位基因的11号染色体缺失或缺失模式与散发性淋巴瘤不同，3)p53和Rb通路中影响细胞周期控制和群体生长和凋亡的关键基因失调模式。使用体外培养的B6.129-Trp53单倍体缺失小鼠的造血干细胞，这些小鼠Cyp2E1基因纯合为零（对苯对苯氧化物的激活至关重要），我们已经能够证明与具有野生型Cyp2E1基因的小鼠相比，多能造血干细胞没有毒性，并且减少了DNA损伤。利用多克隆兔抗s -苯基半胱氨酸，我们正在开发测定骨髓间室苯内合水平的测定方法，以确定剂量与输送到靶组织的剂量之间的关系。这将有助于验证一种假设，即骨髓基质细胞代谢的低剂量苯和局部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发生。

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.