HEPTOCYTE PROLIFERATION AND AFLATOXIN METABOLISM

肝细胞的持续增殖与黄曲霉毒素（AFB1）代谢和肝细胞癌（HCC）发展方面的转基因不成熟表型相关的假设将在p53缺失和p53ser246突变转基因以及HBV小鼠中进行分析。在世界范围内，HCC可以说是导致癌症死亡率最高的肿瘤。世界上高危地区发生的hcc与肝损伤（HBV和HCV）后肝细胞的持续增殖以及黄曲霉毒素（AFB1）暴露有关。AFB1/HBV相关hcc的肿瘤抑制基因p53密码子249 （p53ser249）突变频率很高。最近我们报道了p53缺失小鼠的肝细胞在成年后继续增殖，并且不会随着年龄的增长而变成多倍体。此外，p53ser246转基因小鼠（相当于人的p53ser249）的肝细胞在G1期保持较高的细胞数量。当在出生后第一周给药时，P53 null和p53ser246小鼠对AFB1肝癌的易感性也增加，并且HBV转基因小鼠对AFB1致癌易感性增加，即使在一个月大的时候给药，而同一品系的正常小鼠具有耐药性。我们希望验证这样的假设，即这些小鼠肝脏中细胞状态的变化反映了与成年小鼠致癌代谢物产生增加和HCC发展风险增加相关的低分化表型。在具体的目的1中，我们将通过AFB1环氧物和加合物的形成、谷胱甘肽-s转移酶活性和p450同工酶的分布来检测不同年龄正常和转基因小鼠AFB1的代谢。此外，将从可卡因处理小鼠、新生和成年转基因小鼠的肝脏中分离卵形细胞或小肝细胞，以确定这些细胞中是否存在AFB1的独特代谢。“成熟”肝细胞增殖的影响将使用部分肝切除肝脏的微粒体来确定，并将检查培养的不同分化阶段的肝细胞对AFB1的代谢。在Specific Aim 2中，AFB1将在一个月后（当正常小鼠对癌变不敏感时）施用于p53+/-半合子小鼠、p53-/-缺失小鼠、p53ser246半合子和纯合子转基因小鼠，以及与p53缺失小鼠杂交的p53ser246小鼠F1小鼠，并测定HCC的发生。乙型肝炎相关损伤和乙型肝炎致肝癌的作用。在特异性目的3中，将检测HBV相关损伤和AFB1肝癌在这些小鼠中的作用。

The hypothesis that continued proliferation of hepatocytes is associated with an transgenic immature phenotype in regard to metabolism of aflatoxin (AFB1) and development of hepatocellular carcinoma (HCC) will be analyzed in p53 null and p53ser246 mutant transgenic, and HBV mice. World-wide HCC is arguably the tumor that causes the highest cancer mortality. HCCs occurring in high-risk areas of the world are associated with continued proliferation of liver cells in response to liver injury (HBV and HCV), and with aflatoxin (AFB1) exposure. AFB1/HBV associated HCCs have a high frequency of mutations in the tumor suppressor gene p53 at codon 249 (p53ser249). Recently we reported that hepatocytes of p53 null mice continue to proliferate into adulthood and do not become polyploid with aging. In addition, hepatocytes of p53ser246 transgenic mice (equivalent to p53ser249 of humans) maintain a high number of cells in G1. P53 null and p53ser246 mice also have increased susceptibility to AFB1 hepatocarcinogenesis when the carcinogen is administered during the first week of life, and HBV transgenic mice are susceptible to AFB1 carcinogenesis, even when the carcinogen is administered at one month of age when normal mice of the same strain are resistant. We wish to test the hypothesis that the changes in status of cells in the livers of these mice reflects a less-differentiated phenotype associated with increase production of carcinogenic metabolites and increased risk of HCC development in adult mice. In specific aim 1, the metabolism of AFB1 at different ages of normal and transgenic mice will be examined by AFB1 epoxide and adduct formation, glutathione-s-transferase activity and p450 isoenzyme distribution. In addition, oval cells or small hepatocytes will be isolated from cocaine-treated mice and from neonatal and adult transgenic livers to determine if there is distinctive metabolism of AFB1 in these cells. The effect of proliferation of "mature" hepatocytes will be determined using microsomes from partially hepatectomized livers, and metabolism of AFB1 by cultured liver cells at different stages of differentiation will be examined. In Specific Aim 2, AFB1 will be administered after one month (when normal mice are not susceptible to carcinogenesis) to p53+/- hemizygous mice, to p53-/- null mice, to p53ser246 hemizygous and homozygous transgenic mice, and to F1 mice of p53ser246 mice cross-bred to p53 null mice and the development of HCC determined. the effect Of HBV associated injury and AFB I hepatocarcinogensis in these mice. In Specific Aim 3, the effect of HBV associated injury and AFB1 hepatocarcinogenesis in these mice will be examined.