Paraoxonase--A biomarker of susceptibility to environmental induced diseases

对氧磷酶（PON 1）是一种A-酯酶，已发现在人类中显示底物依赖性多态性。测定个体的PON 1状态（包括PON 1/192基因型和表达水平）可允许使用这种酶作为易感性的生物标志物。由于PON 1解毒许多有机磷和代谢生物活性氧化脂质，PON 1的研究对农药中毒对心血管疾病，特别是动脉粥样硬化的易感性有直接影响。此外，P0 N 1作为“抗氧化剂”蛋白的新兴作用可能在某些神经退行性疾病如帕金森病中具有重要性。该提案的总体目标是进行一系列实验，增加对PON 1功能和调节的重要认识，从而通过更好地定义可能影响其在大型流行病学研究中使用的变量，增加其作为易感性可靠标志物的信心。具体目标是：1。评估环境化学物质对PON 1活性的影响。 我们已经积累了令人信服的证据表明，PON 1的表达水平与基因型一样重要。在单个PO 11基因型中存在至少13倍的酶活性差异。因此，环境化学物质对PON 1活性的调节可能代表了个体PON 1状态的重要性。我们将集中在一些金属，存在于危险废物的网站，并将测量其干扰PON 1活性在体外和体内的能力。通过使用纯化的Arg/192或Gln/192 PON 1，我们还将确定是否任一基因型对这些环境化合物的抑制表现出更高的敏感性。2.确定PON 1的其他底物。我们已经表明，多态性的影响是“逆转”的一些有机磷酸盐底物。因此，我们的第一个目标是评估多态性的影响是“逆转”的一些有机磷酸盐底物。第二个目的是鉴定PON 1对许多有机磷酸酯的其他无毒底物水解能力。第二个目的是鉴定另外的无毒底物，因为迄今为止测试的那些底物，例如苯乙酸盐，没有显示出任何多态性。无毒、多态性底物的鉴定对于大规模流行病学研究和临床环境中使用的高通量检测将是非常有用的。评估PON 1基因型和表达水平对有机磷及其氧合酶毒性和毒代动力学的影响。为了这个目的，我们将集中在二嗪磷和二嗪磷氧磷，和他们的毒代动力学将使用生理为基础的动力学（PBK）模型进行研究。通过将PBK模型中二嗪农和代谢物代谢的总体速率与来自每种小鼠基因型（PON 1 +/+; PON =/-; PON 1-/-; PON 1-/- Arg/192; PON 1-/- Gln/192;后两者是仅表达一种或另一种人类等位基因的转基因小鼠）的每只小鼠的浓度-时间数据拟合，评价PON 1多态性的影响。将比较所得值或峰浓度、血浆浓度-时间曲线下面积、清除率和半衰期，以定量辨别基因型对毒代动力学的影响。此外，各种毒理动力学模型将用于探测二嗪磷和二嗪磷-氧磷的血液和组织浓度与乙酰胆碱酯酶抑制程度之间的关系。4.探讨PON 1状态是否是帕金森病发病的危险因素。几项流行病学研究表明，接触农药可能是帕金森病的一个危险因素，最近的一项研究表明，PON 1/Q192基因型可能是该疾病的一个危险因素。我们建议通过确定对照组和帕金森病患者组中的PON 1状态来确认和扩展这一初步观察结果，这些患者是该超级基金项目中另外两个项目的一部分。

Paraoxonase (PON1) is an A-esterase that has been found to display a substrate-dependent polymorphism in humans. Determination of PON1 status (which includes both PON1/192 genotype and level of expression) of an individual may allow the use of this enzyme as a biomarker of susceptibility. Because PON1 detoxifies a number of organophosphates and metabolizes bioactive oxidized lipids, studies of PON1 have a direct impact on the susceptibility on pesticide poisoning to cardiovascular diseases, particularly atherosclerosis. Furthermore, the emerging role of PON1 as an "antioxidant" protein may have importance in certain neuro- degenerative diseases such as Parkinson's disease. The general aim of this proposal is to carry out a series of experiments that would add important knowledge on PON1 function and modulation, thus increasing confidence in its use as a reliable marker of susceptibility by better defining variables that may effect its use in large epidemiological studies. Specific aims are: 1. To assess the effects of environmental chemicals on PON1 activity. We have been accumulating convincing evidence suggesting that the level of expression of PON1 is as important as the genotype. At least 13-fold differences in enzyme activity exist within a single PO11 genotype. Modulation of PON1 activity by environmental chemicals may, therefore, represent an important of an individual's PON1 status. We will focus on a number of metals, present in hazardous waste sites, and will measure their ability to interfere with PON1 activity in vitro and in vivo. By using purified Arg/192 or Gln/192 PON1, we will also determine whether either genotype displays higher sensitivity to inhibition by these environmental compounds. 2. To identify additional substrates of PON1. We have shown that the effect of the polymorphism is "reversed" for some organophosphate substrates. Our first objective is, therefore, to assess that the effect of the polymorphism is "reversed" for some organophosphate substrates. A second objective is to identify additional non-toxic substrates hydrolyzing ability of PON1 on a number of organophosphates. A second objective is to identify additional non- toxic substrates, as those tested so far, such as phenylacetate, do not show any polymorphism. Identification of non-toxic, polymorphic substrates, will be extremely useful for a high throughput assay to be used in large epidemiological studies and in the clinical setting.3. To assess the role of PON1 genotypes and levels of expression influencing the toxicity and toxicokinetics of organophosphates and their oxons. For this purpose we will focus on diazinon and diazinon-oxon, and their toxicokinetics will be studied using a physiologically-based kinetic (PBK) model. The effects of PON1 polymorphism will be evaluated by fitting the overall rates of diazinon and metabolite metabolism in the PBK model to the concentration-time data from each mouse from each mouse genotype (PON1+/+; PON =/-; PON1 -/-; PON -/- Arg/192; PON1 -/- Gln/192; the latter two are transgenic mice only expressing one or the other of the human alleles). The resulting values or peak concentrations, areas under the plasma concentration-time curves, clearances, and half-lives will be compared in order to quantitatively discern effects of genotype on toxicokinetics. In addition, various toxicodynamics models will be used to probe for relationships between blood and tissue concentrations of diazinon and diazinon-oxon, and the degree of acetylcholinesterase inhibition. 4.To investigate whether PON1 status is a risk factor in the development of Parkinson's disease. Several epidemiological studies have suggested that exposure to pesticides may be a risk factor for Parkinson's disease, and a recent study suggests that the PON1/Q192 genotype may be a risk factor for the disease. We propose to confirm and expand this preliminary observation by determining PON1 status in groups of control and Parkinson's disease patients that are part of two other projects in this Superfund program.