INTERACTIONS: LIPID METABOLISM AND PHTHALATE TOXICITY

在环境中广泛分布的邻苯二甲酸酯， 大鼠肝细胞癌诱生过氧化酶体 目前尚不清楚的机制。有证据表明，体内的氧化应激 已经涉及；然而，过氧化物体过氧化氢的产生 在体内接触其中一些化学物质后不会增加， 对这一解读提出严重质疑。因此，在以下方面存在重要差距 我们的知识是存在的，因此需要对新想法进行评估 在这类重要的化学物质导致 癌症变得清晰起来。我们实验室的主要发现显示，2- 增塑剂邻苯二甲酸二乙基己酯的代谢物乙基己醇 (DEHP)，在门脉周围代谢活跃的区域选择性毒性 解偶联线粒体氧化磷酸化对肝小叶的影响 在体内用强效非遗传毒性药物进行慢性治疗 致癌物WY-14,643在一定条件下引起灌流肝脏解偶联 最终导致肿瘤。基于这些新发现，我们 假设高亲脂性增塑剂和降脂药物 积聚在线粒体膜上，解偶联氧化磷酸化 并改变细胞内能量，导致细胞内 钙。我们进一步提出，钙可提高二十烷类化合物和/或 枯否细胞产生丝裂原细胞因子并增加细胞数量 通过可能涉及蛋白激酶c的机制进行营业额。我们计划 关注Kupffer细胞，因为它们被钙激活，而且因为 我们最近已经证明WY-14,643引起细胞内 钙在培养中的效率比2-乙基己醇高得多。这些 假说将首先通过评估模型化合物是否 DEHP和WY-14,643在慢性暴露后引起氧化应激 通过测量灌流的肝脏中过氧化氢的产生来检测活体。第二， DEHP和WY-14,643在肝脏亚细胞组分中的蓄积 在对大鼠进行不同时间的治疗后，最长可达4个月 体内外氧化磷酸化的测定和解偶联 将会被评估。第三，细胞内游离钙的测定 Kupffer细胞和实质细胞中Fura-2荧光的变化。 此外，还将测量二十烷类化合物和细胞因子的释放。库普弗 然后，细胞将被选择性地用Gd处理来破坏 以检验它们对该序列至关重要的假设 事件的发生，并对导致小区周转率上升负责 由WY-14,643。通过严格测试这些假设，不仅将 需要关于邻苯二甲酸盐和脂类作用机理的信息- 提供降低药物，但将使用的关键信息 评估这类重要的癌症对人类的健康风险 就会产生化学物质。 GRANT=R01HD07503 拟议研究的总体目标是审查监管机构 影响睾丸胚泡有丝分裂后发育的机制 细胞。哺乳动物精子中的主要钙结合蛋白是 卡尔斯佩明。我们已经克隆了该蛋白的编码基因。分析 对该基因的测序和分析表明，钙精蛋白来源于一种 转录单位，也编码一种新的钙-钙调蛋白依赖 蛋白激酶。该基因也已被克隆和测序。鉴于 该蛋白在早期初级精母细胞中首次被检测到，并且 在最后一次减数分裂后下降到较低水平 MRNA首先在粗线期初级精母细胞中表达，并持续表达 随着细胞完成减数分裂并经历末期而增加 差异化。我们已经分离出基因组DNA来编码整个 转录单位，并建议表征其结构 基因的组织。然后我们将利用转基因的哺乳动物 细胞，睾丸转录系统和转基因小鼠来确定 控制细胞特异性mRNAs表达的分子机制。这个 假设是另一种转录起始，然后是一个 所需独特的RNA加工事件参与了钙精蛋白 制作。表达载体已被用来生产钙精蛋白和 以确定功能和/或所需的关键氨基酸 对活动的监管。一种构成激酶已经被用来开发 以受调控的方式表达这种酶的哺乳动物细胞系 鉴定被转录因子反式激活的DNA元件 活性酶。我们建议将这种机制描述为 该激酶的过表达会导致G2期细胞周期受阻 对该激酶有反应的转录因子。转基因小鼠将成为 用来评估改变浓度的后果 蛋白激酶对精子发生和精子活力的影响。不同的推动者 将用于更改时间表达式和/或 生殖细胞发育过程中的蛋白质。我们预测，该激酶将 在减数分裂的开始、进展或完成中起作用，而 钙精蛋白将参与精子细胞的终末分化 或者精子的能动性。

Phthalic acid esters, which are widely dispersed in the environment, induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. Evidence that oxidative stress in involved has been presented; however, peroxisomal H2O2 generation does not increase in vivo following exposure to some of these chemicals, seriously questioning this interpretation. Therefore, important gaps in our knowledge exist and it follows that new ideas need to be evaluated before the mechanisms by which this important class of chemicals causes cancer become clear. Key findings from our laboratory showed that 2- ethylhexanol, a metabolite of the plasticizer di(ethylhexyl) phthalate (DEHP), was selectively toxic in metabolically active periportal regions of the liver lobule by uncoupling mitochondrial oxidative phosphorylation and that chronic treatment in vivo with the potent nongenotoxic carcinogen WY-14,643 caused uncoupling in perfused liver under conditions which lead ultimately to tumors. Based on these new findings, we postulate that highly lipophilic plasticizers and lipid-lowering drugs accumulate in mitochondrial membranes, uncouple oxidative phosphorylation and alter cellular energetics leading to increases in intracellular calcium. We further propose that calcium elevates eicosanoid and/or mitogenic cytokine production by Kupffer cells and increases cell turnover by mechanisms possible involving protein kinase c. We plan to focus on Kupffer cells because they are activated by calcium and because we have shown very recently that WY-14,643 cause elevate intracellular calcium much more efficiently that 2-ethylhexanol in culture. These hypotheses will be tested first by assessing whether the model compounds DEHP and WY-14,643 cause oxidative stress following chronic exposure in vivo by measuring H2O2 production in the perfused liver. Second, the accumulation of DEHP and WY-14,643 in hepatic subcellular fractions following treatment of rats for various times up to four months will be measured and uncoupling of oxidative phosphorylation in vitro and in vivo will be assessed. Third, intracellular free calcium will be determined from changes in FURA-2 fluorescence in Kupffer and parenchymal cells. In addition, eicosanoid and cytokine release will be measured. Kupffer cells will then be destroyed selectively by treatment with gadolinium chloride to test the hypothesis that they are critical to this sequence of events and are responsible for the elevation in cell turnover caused by WY-14,643. By testing these hypotheses rigorously, not only will much needed information on the mechanism of action of phthalates and lipid- lowering drugs be provided, but critical information which will be used to assess the health risk posed to humans by this important class of chemicals will be generated. GRANT=R01HD07503 The overall goals of the proposed research are to examine regulatory mechanisms that influence post-mitotic development of testicular germ cells. The major calcium binding protein in mammalian sperm is calspermin. We have cloned the cDNA encoding this protein. Analysis of the sequence and of the gene revealed that calspermin is derived from a transcription unit that also encodes a novel calcium-calmodulin dependent protein kinase. This cDNA has also been cloned and sequenced. Whereas the kinase mRNA is first detected in early primary spermatocytes and declines to a ow level following the last meiotic division, calspermin mRNA is first expressed in pachytene primary spermatocytes and continues to increase as cell complete meiosis and undergo terminal differentiation. We have isolated genomic DNA that encodes the entire transcription unit and propose to characterize the structural organization of the gene. We will then utilize transfected mammalian cells, a testicular transcription system and transgenic mice to determine the molecular mechanisms that control cell specific mRNA expression. The hypothesis is that alternative transcriptional initiation followed by a requisite unique RNA processing event are involved in calspermin production. Expression vectors have been used to produce calspermin and the kinase and to identify key amino acids required for function and/or regulation of activity. A constitutive kinase has been used to develop mammalian cell lines that express the enzyme in a regulated fashion and to identify a transcriptional DNA element that is trans-activated by the active enzyme. We propose to characterize the mechanism by which the overexpression of the kinase results in a G2 cell cycle block and the transcription factor responsive to the kinase. Transgenic mice will be utilized to evaluate the consequences of altering the concentration of the kinase on spermatogenesis and sperm motility. Different promoters will be employed to change the temporal expression and/or amount of the proteins during germ cell development. We predict that the kinase will play a role in the onset, progression or completion of meiosis whereas calspermin will be involved in terminal differentiation of the spermatid or sperm motility.