HEPTOCYTE PROLIFERATION AND AFLATOXIN METABOLISM

关于肝细胞持续增殖与黄曲霉毒素(AFB1)代谢和肝细胞癌(HCC)发生相关的转基因未成熟表型的假设，将在p53缺失和p53ser246突变的转基因和乙肝小鼠中进行分析。全球范围内的肝癌可以说是导致最高癌症死亡率的肿瘤。发生在世界高危地区的HCCs与肝损伤(乙肝病毒和丙型肝炎病毒)和黄曲霉毒素(AFB1)暴露导致的肝细胞持续增殖有关。AFB1/HBV型肝细胞癌的抑癌基因p53在249密码子(P53ser249)上有很高的突变频率。最近，我们报道了P53基因缺失小鼠的肝细胞继续增殖到成年期，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(相当于人类的p53ser249)的肝细胞维持在G1期的大量细胞。当在生命的第一周内注射致癌物时，p53ser246和p53ser246小鼠也增加了对AFB1肝癌的易感性，即使在同一品系的正常小鼠具有抵抗力的一个月大时注射致癌剂，HBV转基因小鼠也容易发生AFB1癌。我们希望验证这一假设，即这些小鼠肝脏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肝癌发展风险增加相关的较低分化表型。具体目标1，通过黄曲霉毒素B_1的环氧化物和加合物的形成、谷胱甘肽-S转移酶活性和P450同工酶的分布来检测黄曲霉毒素B_1在正常和转基因小鼠不同日龄的代谢。此外，将从可卡因处理的小鼠以及新生和成年转基因肝脏中分离出卵圆形细胞或小肝细胞，以确定这些细胞中是否存在AFB1的独特新陈代谢。将使用部分切除肝脏的微粒体来确定成熟肝细胞的增殖效果，并将检测不同分化阶段培养的肝细胞对黄曲霉毒素B 1的代谢。在特定目的2中，将黄曲霉毒素B_1在1个月后(在正常小鼠不易致癌的情况下)注射给p53+/-半合子小鼠、p53-/-缺失型小鼠、p53ser246半合子和纯合子转基因小鼠，以及p53ser246小鼠与p53缺失型小鼠杂交的F1小鼠，并测定其肝癌的发生情况。乙肝病毒相关损伤及黄曲霉毒素B I在小鼠肝癌发生中的作用在具体目标3中，将检测这些小鼠的乙肝病毒相关损伤和黄曲霉毒素B_1(AFB1)肝癌发生的影响。

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.