用于测量环境和实验室产生的气溶胶诱导的氧化应激的细胞和细胞测定法。

N L Ng, W Y Tuet, Y Chen, S Fok, D Gao, M S Tagle Rodriguez, M Klein, A Grosberg, R J Weber, J A Champion
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Many prior studies have focused on the aerosol of primary origin (e.g., the aerosol emitted from combustion engines) although the aerosol formed from the oxidation of volatile species, secondary organic aerosol (SOA), has been shown to be the predominant type of aerosol even in urban areas. Current SOA health studies are limited in number, and as such, the health effects of SOA are poorly characterized. Also, there is a lack of perspective in terms of the relative toxicities of different SOA systems. Additionally, although chemical assays have identified some SOA constituents associated with adverse health endpoints, the applicability of these results to cellular responses has not been well established.</p><p><strong>Specific aims: </strong>The overall objective of this study was to better understand the oxidative properties of different types and components of PM mixtures (especially SOA) through systematic laboratory chamber experiments and ambient field studies. 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A suite of instruments was utilized to monitor gas- and particle-phase species. Bulk aerosol properties (e.g., O:C, H:C, and N:C ratios) were measured using a high-resolution time-of-flight aerosol mass spectrometer. Filter samples were collected for chemical oxidative potential and cellular measurements. For the naphthalene system, multiple filter samples were collected over the course of a single experiment to collect aerosols of different photochemical aging.</p><p><p>For all filter samples, chemical oxidative potentials were determined for water-soluble extracts using a semiautomated DTT assay system. Murine alveolar macrophages and neonatal rat ventricular myocytes were also exposed to PM samples extracted in cell culture medium to investigate cellular responses. 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The presence of iron sulfate seed particles did not have an apparent effect on oxidative potentials; however, a higher level of ROS/RNS production was observed for all SOA formed in the presence of iron sulfate compared with ammonium sulfate. We also identified a significant positive correlation between ROS/RNS production and average carbon oxidation state, a bulk aerosol property. It may therefore be possible to roughly estimate ROS/RNS production using this property, which is readily obtainable. This correlation may have significant implications as aerosols have an atmospheric lifetime of a week, during which average carbon oxidation state increases because of atmospheric photochemical aging. Our results suggest that aerosols might become more toxic as they age in the atmosphere. Finally, in the context of ambient samples, laboratory-generated SOA induced comparable or higher levels of ROS/RNS. 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引用次数: 0

摘要

导言:许多研究证实,暴露于空气污染或大气颗粒物(PM)与不良健康影响之间存在关联。越来越多的研究表明,氧化应激是可吸入颗粒物诱发健康影响的可能机制,因此,人们开发了许多化学和细胞检测方法来研究可吸入颗粒物诱发的氧化剂生成。尽管近年来取得了重大进展,但这一研究领域仍有许多空白尚未填补。之前的许多研究都侧重于原生气溶胶(如内燃机排放的气溶胶),但挥发性物质氧化形成的气溶胶,即二次有机气溶胶(SOA),已被证明是气溶胶的主要类型,即使在城市地区也是如此。目前的 SOA 健康研究数量有限,因此,SOA 对健康的影响尚不明确。此外,人们对不同 SOA 系统的相对毒性也缺乏认识。此外,虽然化学分析已经确定了一些与不利健康终点相关的 SOA 成分,但这些结果对细胞反应的适用性尚未得到很好的确定:本研究的总体目标是通过系统的实验室室实验和环境实地研究,更好地了解不同类型和成分的可吸入颗粒物混合物(尤其是 SOA)的氧化特性。2 确定与 ROS/RNS 生成相关的环境 PM 成分,并评估化学检测结果是否代表了细胞在 ROS/RNS 生成方面的反应。研究常见生物前体和人为前体在不同条件下(如湿度、氮氧化物[NOx]和氧化还原活性金属)形成的 SOA 的相对毒性,并提供相关视角,同时确定与细胞反应相关的大量气溶胶特性:作为东南部空气污染与流行病学中心(SCAPE)研究的一部分,2012 年 6 月至 2013 年 10 月期间从大亚特兰大地区的城市和农村地点收集了环境 PM 样品。水溶性物质(如水溶性有机碳 [WSOC]、褐碳 [Br C] 和金属)的浓度使用各种仪器进行表征。实验室研究是在佐治亚理工学院环境室 (GTEC) 中进行的,目的是在控制良好的光氧化条件下产生 SOA。生物源前体(异戊二烯、α-蒎烯和β-石竹烯)和人为源前体(十五烷、间二甲苯和萘)在不同的形成条件(干燥与潮湿、氮氧化物、硫酸铵与硫酸铁种子颗粒)下被氧化,产生不同化学成分和质量负荷的 SOA。对于萘系统,进行了一系列不同初始碳氢化合物浓度的实验,以产生不同氧化程度的气溶胶。利用一套仪器来监测气相和颗粒相物种。使用高分辨率飞行时间气溶胶质谱仪测量了气溶胶的总体特性(如 O:C、H:C 和 N:C 比率)。收集过滤器样本用于化学氧化潜能和细胞测量。对于萘系统,在一次实验过程中收集多个过滤器样本,以收集不同光化学老化程度的气溶胶。对于所有过滤器样本,使用半自动 DTT 分析系统测定水溶性提取物的化学氧化电位。小鼠肺泡巨噬细胞和新生大鼠心室肌细胞也暴露于在细胞培养基中提取的 PM 样品,以研究细胞反应。使用细胞内 ROS/RNS 探针羧基-2',7'-二氯二氢荧光素二乙酸酯(羧基-H2DCFA)检测 ROS/RNS 的产生,而使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT)评估细胞代谢活性。最后,使用酶联免疫吸附试验(ELISA)测量暴露后细胞因子的产生,即肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的分泌水平。为了确定与氧化特性相关的可吸入颗粒物成分,对氧化特性(细胞反应或 DTT 活性)和气溶胶成分(金属、元素比率等)进行了线性回归。 结果:我们优化了 ROS/RNS 检测的几个参数,包括细胞密度(巨噬细胞为 2 × 104 个细胞/孔,心肌细胞为 3 33 × 104 个细胞/孔)和探针浓度(10 µM):我们优化了 ROS/RNS 检测的几个参数,包括细胞密度(巨噬细胞为 2 × 104 个细胞/孔,心肌细胞为 3.33 × 104 个细胞/孔)、探针浓度(10 µM)和样品培养时间(24 小时)。环境气溶胶和实验室产生的气溶胶的研究结果表明,ROS/RNS 的产生高度依赖于剂量,并且与 PM 剂量呈非线性关系。在本研究调查的剂量-反应指标(最大反应、反应比基线高 10%的剂量[阈值]、达到 50%反应的剂量[EC50]、达到最大反应的速率[希尔斜率]和剂量-反应曲线下面积[AUC])中,我们发现 AUC 是最可靠的参数,其信息量与剂量范围无关。在夏季采集的环境样本中,AUC 所代表的 ROS/RNS 产量与 DTT 所测量的化学氧化潜能之间存在明显的正相关。相反,在冬季采集的环境样本中,无论相应的 DTT 活性如何,都能观察到相对稳定的 AUC。我们还确定了与夏季样本 AUC 显著相关的几种可吸入颗粒物成分(WSOC、BrC、铁和钛)。有机物与 ROS/RNS 生成之间的强相关性突出表明,有必要了解有机气溶胶对可吸入颗粒物引起的健康影响的贡献。对于实验室产生的气溶胶,前体特性对氧化潜能有很大影响,异戊二烯和萘的 SOA 的 DTT 活性分别最低和最高。前体特征和形成条件都会对接触 SOA 后诱发的炎症反应产生重大影响,而且对于光氧化产物具有相似碳链长度和功能的 SOA 前体,还能确定几种反应模式。硫酸铁种子颗粒的存在对氧化电位没有明显的影响;但是,与硫酸铵相比,在硫酸铁存在下形成的所有 SOA 产生的 ROS/RNS 水平都更高。我们还发现,ROS/RNS 的产生与平均碳氧化态(气溶胶的一种基本特性)之间存在明显的正相关关系。因此,可以利用这一容易获得的特性来粗略估计 ROS/RNS 的产生。气溶胶在大气中的寿命为一周,在此期间,由于大气光化学老化,平均碳氧化态会增加,因此这种相关性可能具有重要意义。我们的研究结果表明,随着气溶胶在大气中的老化,其毒性可能会增加。最后,与环境样本相比,实验室产生的 SOA 诱导的 ROS/RNS 水平相当或更高。除萘的氧化电位较高外,所有实验室 SOA 系统的氧化电位都与在环境样本中观察到的氧化电位相当。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Cellular and Acellular Assays for Measuring Oxidative Stress Induced by Ambient and Laboratory-Generated Aerosols.

Cellular and Acellular Assays for Measuring Oxidative Stress Induced by Ambient and Laboratory-Generated Aerosols.

Cellular and Acellular Assays for Measuring Oxidative Stress Induced by Ambient and Laboratory-Generated Aerosols.

Introduction: Many studies have established associations between exposure to air pollution, or atmospheric particulate matter (PM), and adverse health effects. An increasing array of studies have suggested oxidative stress as a possible mechanism by which PM-induced health effects arise, and as a result, many chemical and cellular assays have been developed to study PM-induced oxidant production. Although significant progress has been made in recent years, there are still many gaps in this area of research that have not been addressed. Many prior studies have focused on the aerosol of primary origin (e.g., the aerosol emitted from combustion engines) although the aerosol formed from the oxidation of volatile species, secondary organic aerosol (SOA), has been shown to be the predominant type of aerosol even in urban areas. Current SOA health studies are limited in number, and as such, the health effects of SOA are poorly characterized. Also, there is a lack of perspective in terms of the relative toxicities of different SOA systems. Additionally, although chemical assays have identified some SOA constituents associated with adverse health endpoints, the applicability of these results to cellular responses has not been well established.

Specific aims: The overall objective of this study was to better understand the oxidative properties of different types and components of PM mixtures (especially SOA) through systematic laboratory chamber experiments and ambient field studies. The study had four specific aims.

1 To develop a cellular assay optimized for measuring reactive oxygen and nitrogen species (ROS/RNS) production resulting from PM exposure and to identify a robust parameter that could represent ROS/RNS levels for comparison with different endpoints.

2 To identify ambient PM components associated with ROS/RNS production and evaluate whether results from chemical assays represented cellular responses in terms of ROS/RNS production.

3 To investigate and provide perspective on the relative toxicities of SOA formed from common biogenic and anthropogenic precursors under different conditions (e.g., humidity, nitrogen oxides [NOx], and redox-active metals) and identify bulk aerosol properties associated with cellular responses.

4 To investigate the effects of photochemical aging on aerosol toxicity.

Methods: Ambient PM samples were collected from urban and rural sites in the greater Atlanta area as part of the Southeastern Center for Air Pollution and Epidemiology (SCAPE) study between June 2012 and October 2013. The concentrations of water-soluble species (e.g., water-soluble organic carbon [WSOC], brown carbon [Br C], and metals) were characterized using a variety of instruments. Samples for this study were chosen to span the observed range of dithiothreitol (DTT) activities.

Laboratory studies were conducted in the Georgia Tech Environmental Chamber (GTEC) facility in order to generate SOA under well-controlled photooxidation conditions. Precursors of biogenic origin (isoprene, α-pinene, and β-caryophyllene) and anthropogenic origin (pentadecane, m-xylene, and naphthalene) were oxidized under various formation conditions (dry vs. humid, NOx, and ammonium sulfate vs. iron sulfate seed particles) to produce SOA of differing chemical composition and mass loading. For the naphthalene system, a series of experiments were conducted with different initial hydrocarbon concentrations to produce aerosols with various degree of oxidation. A suite of instruments was utilized to monitor gas- and particle-phase species. Bulk aerosol properties (e.g., O:C, H:C, and N:C ratios) were measured using a high-resolution time-of-flight aerosol mass spectrometer. Filter samples were collected for chemical oxidative potential and cellular measurements. For the naphthalene system, multiple filter samples were collected over the course of a single experiment to collect aerosols of different photochemical aging.

For all filter samples, chemical oxidative potentials were determined for water-soluble extracts using a semiautomated DTT assay system. Murine alveolar macrophages and neonatal rat ventricular myocytes were also exposed to PM samples extracted in cell culture medium to investigate cellular responses. ROS/RNS production was detected using the intracellular ROS/RNS probe, carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFA), whereas cellular metabolic activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Finally, cytokine production, that is, secreted levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were measured post-exposure using an enzyme-linked immunosorbent assay (ELISA). To identify PM constituents associated with oxidative properties, linear regressions between oxidative properties (cellular responses or DTT activity) and aerosol composition (metals, elemental ratios, etc.) were evaluated using Pearson's correlation coefficient, where the significance was determined using multiple imputation and evaluated using a 95% confidence interval.

Results: We optimized several parameters for the ROS/RNS assay, including cell density (2 × 104 cells/well for macrophages and 3.33 × 104 cells/well for cardiomyocytes), probe concentration (10 µM), and sample incubation time (24 hours). Results from both ambient and laboratory-generated aerosols demonstrate that ROS/RNS production was highly dose-dependent and nonlinear with respect to PM dose. Of the dose-response metrics investigated in this study (maximum response, dose at which the response is 10% above the baseline [threshold], dose at which 50% of the response is attained [EC50], rate at which the maximum response is attained [Hill slope], and area under the dose-response curve [AUC]), we found that the AUC was the most robust parameter whose informativeness did not depend on dose range.

A positive, significant correlation was observed between ROS/RNS production as represented by AUC and chemical oxidative potential as measured by DTT for ambient samples collected in summer. Conversely, a relatively constant AUC was observed for ambient samples collected in winter regardless of the corresponding DTT activity. We also identified several PM constituents (WSOC, BrC, iron, and titanium) that were significantly correlated with AUC for summer samples. The strong correlation between organic species and ROS/RNS production highlights a need to understand the contribution of organic aerosols to PM-induced health effects. No significant correlations were observed for other ROS/RNS metrics or PM constituents, and no spatial trends were observed.

For laboratory-generated aerosol, precursor identity influenced oxidative potentials significantly, with isoprene and naphthalene SOA having the lowest and highest DTT activities, respectively. Both precursor identity and formation condition significantly influenced inflammatory responses induced by SOA exposure, and several response patterns were identified for SOA precursors whose photooxidation products share similar carbon-chain length and functionalities. The presence of iron sulfate seed particles did not have an apparent effect on oxidative potentials; however, a higher level of ROS/RNS production was observed for all SOA formed in the presence of iron sulfate compared with ammonium sulfate. We also identified a significant positive correlation between ROS/RNS production and average carbon oxidation state, a bulk aerosol property. It may therefore be possible to roughly estimate ROS/RNS production using this property, which is readily obtainable. This correlation may have significant implications as aerosols have an atmospheric lifetime of a week, during which average carbon oxidation state increases because of atmospheric photochemical aging. Our results suggest that aerosols might become more toxic as they age in the atmosphere. Finally, in the context of ambient samples, laboratory-generated SOA induced comparable or higher levels of ROS/RNS. Oxidative potentials for all laboratory SOA systems, with the exception of naphthalene (which was higher), were all comparable with oxidative potentials observed in ambient samples.

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