HEPTOCYTE PROLIFERATION AND AFLATOXIN METABOLISM

肝细胞的持续增殖与黄曲霉毒素（AFB 1）代谢和肝细胞癌（HCC）发生的转基因未成熟表型相关的假设将在p53 null和p53 ser 246突变转基因小鼠和HBV小鼠中进行分析。 在世界范围内，HCC可以说是导致最高癌症死亡率的肿瘤。 世界上高风险地区发生的HCC与肝细胞对肝损伤（HBV和HCV）的持续增殖以及黄曲霉毒素（AFB 1）暴露有关。 AFB 1/HBV相关的HCC具有高频率的抑癌基因p53第249位密码子突变（p53 ser 249）。 最近，我们报道了p53基因敲除小鼠的肝细胞继续增殖到成年期，并没有成为多倍体随着年龄的增长。 此外，p53 ser 246转基因小鼠（相当于人的p53 ser 249）的肝细胞在G1期维持大量细胞。 当在出生后第一周内给予致癌物时，P53 null和p53 ser 246小鼠对AFB 1肝癌发生的易感性也增加，而HBV转基因小鼠对AFB 1肝癌发生易感，即使当同一品系的正常小鼠在一个月龄时给予致癌物时也是如此。 我们希望检验这样一个假设，即这些小鼠肝脏中细胞状态的变化反映了与致癌代谢物产生增加和成年小鼠HCC发展风险增加相关的分化程度较低的表型。 在具体目标1中，将通过AFB 1环氧化物和加合物形成、谷胱甘肽-S-转移酶活性和p450同工酶分布来检测正常和转基因小鼠不同年龄的AFB 1代谢。 此外，将从可卡因处理的小鼠以及新生儿和成年转基因肝脏中分离卵圆细胞或小肝细胞，以确定这些细胞中是否存在AFB 1的独特代谢。 将使用来自部分肝切除肝脏的微粒体确定“成熟”肝细胞增殖的影响，并将检查不同分化阶段的培养肝细胞对AFB 1的代谢。 在特定目标2中，将在一个月后（当正常小鼠对致癌作用不敏感时）对p53+/-半合子小鼠、p53-/-缺失小鼠、p53 ser 246半合子和纯合子转基因小鼠以及p53 ser 246小鼠与p53缺失小鼠杂交的F1小鼠给予AFB 1，并确定HCC的发生。HBV相关性损伤和AFB-I致小鼠肝癌的作用。 在具体目标3中，将检查这些小鼠中HBV相关损伤和AFB 1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.