HEPATOTOXICITY OF ATRAZINE AND ITS DEGRADATION PRODUCTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 阿特拉津是世界上使用最多的除草剂之一。本研究的重点是研究阿特拉津及其降解产物的肝毒性。在6/06-5/07的资助期内，我们利用高效液相-电喷雾质谱/质谱法研究了阿特拉津在铁(III)存在或不存在的情况下在大鼠肝微球体内的代谢。孵育12h后，用三氯乙酸+50%甲醇提取代谢产物。无论有没有Fe(III)存在，总能检测到四种I相代谢产物，即去乙基阿特拉津、去异丙基阿特拉津、羟基阿特拉津和去异丙基羟基阿特拉津。去乙基阿特拉津和去异丙基阿特拉津的浓度接近。比羟基阿特拉津和去异丙基羟基阿特拉津高50倍。在Fe(III)存在的情况下，在孵育液中发现了另一种代谢物，即二去烷基阿特拉津。双烷基特拉津的浓度随着Fe(III)浓度的增加而增加。据我们所知，这是首次在这个生物体外系统中研究阿特拉津的降解产物。此外，我们还建立了一种检测脂质过氧化的生物标志物4-羟基-壬烯醛(4-HNE)的高效液相色谱-MS/MS联用方法。该方法选择性高，灵敏度高，在大鼠肝微粒体孵育液中的检出限为10 ng/mL4-HNE。这一组的三家同行审查的出版物在这一资助期承认了RCMI的赠款(12RR13459)。明年，我们计划研究已确定的阿特拉津降解产物对NADPH诱导的大鼠肝微粒体脂质过氧化的影响，并将结果与阿特拉津的结果进行比较，从而获得这些降解产物对其母体化合物阿特拉津的相对毒性。4-HNE的TBAS分析和有利的高效液相-MS/MS分析将用于评估氧化应激状态。此外，还将使用包括半经验AM1在内的计算方法探索潜在的结构-活性关系(SAR)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Atrazine is one of the herbicides that are most heavily used in the world. The proposed research is focused on studying the hepatotoxicity of atrazine and its degradation products. In the funding period from 6/06 to 5/07, we studied the metabolism of atrazine in rat liver microsome with or without the presence of iron (III) by using an advantageous HPLC-ESI-MS/MS method. After incubation for 12 hours, metabolites were extracted by trichloroacetic acid + 50% methanol. Four phase I metabolites, i.e. deethylatrazine, deisopropylatrazine, hydroxyatrazine, and deisopropylhydroxyatrazine were always detected no matter Fe(III) presented or not. The concentrations of deethylatrazine and deisopropylatrazine were approx. 50-times higher than those of hydroxyatrazine and deisopropylhydroxyatrazine. In the presence of Fe(III), another metabolite, i.e. didealkylatrazine was identified in the incubation solutions. The concentration of didealkylatrazine was found to increase with the increase in Fe(III) concentration. To the best of our knowledge, this was the first study of atrazine degradation products in this biological in vitro system. In addition, we developed an HPLC-MS/MS method for the determination of 4-hydroxy-nonenal (4-HNE), a specific biomarker for lipid peroxidation. The method was highly selective and very sensitive with an LOD of 10 ng /mL 4-HNE in rat liver microsome incubation solution. Three peer-reviewed publications from this group are acknowledging the RCMI grant (12RR13459) in this funding period. Next year, we plan to study the effects of the identified degradation products of atrazine on NADPH induced lipid peroxidation in rat liver microsome, and compare the results with those from atrazine, thus obtaining the relative toxicity of the degradation products against their parent compound, atrazine. Both TBAS assay and the advantageous HPLC-MS/MS assay for 4-HNE will be used to assess the oxidative stress status. In addition, the potential structure-activity relationship (SAR) will be explored using computational methods including the semi empirical AM1.