Leukemogenesis In Genetically-altered Mouse Models

由于电离辐射和苯（一种拟辐射物质）有可能诱发造血系统癌症，特别是在非常年轻的人中，因此公众对它们的健康关注有所增加。早期暴露于辐射和苯可能与导致间变性贫血的血液形成元素的抑制有关，随后是淋巴瘤或骨髓增生异常表型，最终可能导致白血病或淋巴瘤。苯的多种暴露途径（吸入、经口和/或皮肤）可能导致递送速率较慢，并导致敏感靶组织的内部剂量较大。在同等吸入和/或经口剂量（mg/kg/体重）下，经口给药后未代谢的苯可能比吸入（14%）多（60%）。选择动物模型实验研究的剂量最关键的是确定解毒途径饱和的暴露水平，并改变递送到造血干细胞区室（例如骨髓）的剂量。对于小鼠吸入暴露，这约为200 ppm（6 h TWA）。在5至50 ppm苯（6小时TWA）之间，尿液代谢物之间没有显着差异。现有数据还表明，吸入暴露于50 ppm（6 h TWA）后，小鼠和人类之间的对苯二酚或粘康酸与苯酚的比值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，苯诱导的造血系统疾病的暴露模型必须考虑苯代谢途径的饱和度、暴露的现有数据以及导致最具血液毒性中间体的高亲和力、低容量代谢途径。我们已经能够表明，B6.129-Trp 53单倍不足的小鼠暴露于间歇性低水平（100 ppm，6 h TWA）迅速发展胸腺淋巴瘤，而小鼠暴露于更频繁和更高水平的苯（200 ppm，6 h TWA）发展肿瘤的速度较慢，发病率显着降低。在这些研究中，苯诱导的p53缺陷小鼠淋巴瘤为：1）克隆（T细胞受体重排是常见的），2）显示携带与散发性淋巴瘤不同的p53野生型等位基因的11号染色体丢失或缺失的模式，和3）显示了p53和Rb通路中关键基因的失调模式，其影响细胞周期控制和群体生长和凋亡。使用B6.129-Trp 53单倍体不足小鼠的造血干细胞培养物，这些小鼠是Cyp 2 E1基因（对苯活化为苯氧化物至关重要）纯合无效的，我们已经能够显示与野生型Cyp 2 E1基因小鼠相比，对多能造血干细胞没有毒性和DNA损伤减少。使用多克隆兔抗-S-苯基半胱氨酸，我们正在开发测定方法，以确定苯加合到骨髓室的水平，从而确定剂量与递送到靶组织的剂量之间的关系。这将有助于检验以下假设，即骨髓基质细胞代谢的较低剂量的苯和局部产生的毒性代谢物可能直接作用于造血干细胞区室并引发肿瘤发生。

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.