Paraoxonase--A biomarker of susceptibility to environmental induced diseases

对氧磷酶(PON1)是一种A-酯酶，在人类中表现出底物依赖的多态。确定个体的PON1状态(包括PON1/192基因和表达水平)可能允许使用这种酶作为易感性的生物标记物。由于PON1能解毒并代谢生物活性氧化脂质，因此对PON1的研究直接影响农药中毒对心血管疾病，特别是动脉粥样硬化的易感性。此外，PON1作为一种“抗氧化剂”蛋白的新角色可能在某些神经退行性疾病中具有重要作用，例如帕金森氏症。这项建议的总体目标是进行一系列实验，以增加关于PON1功能和调节的重要知识，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，增加使用PON1作为可靠的易感性标记的信心。具体目标是：1.评估环境化学品对PON1活性的影响。我们一直在积累令人信服的证据，表明PON1的表达水平与基因同样重要。在同一个PO11基因中，酶活性至少存在13倍的差异。因此，环境化学品对PON1活性的调节可能代表着一个人的PON1状态的一个重要方面。我们将重点关注危险废物场所中存在的一些金属，并将测量它们在体外和体内干扰PON1活性的能力。通过使用纯化的Arg/192或Gln/192 PON1，我们还将确定这两种基因型中的哪一种对这些环境化合物的抑制表现出更高的敏感性。2.鉴定PON1的额外底物。我们已经证明，对于某些有机磷底物来说，这种多态的作用是“相反的”。因此，我们的第一个目标是评估这种多态对某些有机磷底物的影响是“反向的”。第二个目标是确定其他无毒底物对PON1在一些有机磷上的水解能力。第二个目标是确定更多的无毒底物，因为到目前为止测试的那些底物，如苯乙酸酯，没有表现出任何多态。鉴定无毒的、多态的底物，对于在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PON1基因类型和表达水平对有机磷及其氧化合物的毒性和毒代动力学的影响。为此，我们将重点关注二氮磷和二氮酮-氧酮，并将使用基于生理的动力学(PBK)模型来研究它们的毒代动力学。通过将PBK模型中二氮磷和代谢物代谢的总体速率与来自每只小鼠的每只小鼠的浓度-时间数据(PON1+/+；PON=/-；PON1-/-；PON-/-Arg/192；PON1-/-Gln/192；后两者是仅表达人类等位基因中的一种或另一种的转基因小鼠)进行匹配，来评估PON1多态的影响。结果值或峰值浓度、血浆浓度-时间曲线下的面积、清除量和半衰期将被比较，以便定量辨别基因对毒代动力学的影响。此外，各种毒理动力学模型将被用来探索二氮农和二氮酮-氧酮的血液和组织浓度之间的关系，以及乙酰胆碱酯酶抑制的程度。4.探讨PON1状态是否为帕金森病发病的危险因素。多项流行病学研究表明，接触杀虫剂可能是帕金森氏症的风险因素，最近的一项研究表明，PON1/Q192基因可能是该疾病的风险因素。我们建议通过确定控制组和帕金森氏病患者的PON1状态来证实和扩大这一初步观察结果，这两组患者是该超级基金计划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.