The effect of titanium dioxide (TiO2) nano-objects, and their aggregates and agglomerates greater than 100 nm (NOAA) on yeast under UV irradiation

Today, nanoparticles are used in various commercial products. One of the most common nanoparticles is titanium dioxide (TiO2). It has a catalytic activity and UV absorption (λ<400 nm), and it generates reactive oxygen species (ROS). The catalytic activity of TiO2 nanoparticle is capable of killing a wide range of microorganisms. In the environment, nanoparticles form structures consisting of primary particles, and their aggregates and agglomerates. These compounds are defined NOAA (nano-objects, and their aggregates and agglomerates greater than 100 nm). The unique properties of TiO2 nanoparticles can be maintained in the environment, thus, the growing use of TiO2 nanoparticles is raising concerns about the environmental risks. The assessment of biological and ecological effects of TiO2-NOAA is necessary. In our previous study, we assessed the effect of TiO2-NOAA on microbes by using Saccharomyces cerevisiae and Escherichia coli. It was shown that TiO2-NOAA decomposed methylene blue under UV irradiation. It suggested that TiO2-NOAA generated ROS under UV irradiation. However, TiO2-NOAA did not show growth inhibition in minimal agar medium under UV irradiation. By adding TiO2-NOAA in medium, colony formation was observed with UV intensity that inactivates microbes. Moreover, TiO2-NOAA adsorbed microbes. These results suggested that the amount of ROS generated by TiO2-NOAA was not enough to inactivate microbes, and TiO2-NOAA might protect microbes from UV. In this study, we assessed the effect of TiO2-NOAA in more detail by using S. cerevisiae. We used DNA microarray analysis for qualitative assessment. Further, we carried out quantitative assessment by using Real Time RT-PCR method for characteristic genes in DNA microarray analysis. To compare yeast cells in various conditions, six kinds of treatment conditions were prepared (Condition 1. adsorbed fraction to TiO2-NOAA under UV, 2. non-adsorbed fraction to TiO2-NOAA under UV, 3. adsorbed fraction to TiO2-NOAA without UV, 4. non-adsorbed fraction to TiO2-NOAA without UV, 5. irradiated UV and 6. negative control). From the result of DNA microarray analysis, the most number of genes was altered in Condition 1, followed by Condition 3 and 5. The genes related to oxidative stress, and the genes related to synthesis of trehalose and glycogen were significantly up-regulated of yeast cells in Condition 1 and 5, and Condition 1 and 3, respectively. These results suggest that yeast cells suffer oxidative stress by TiO2-NOAA under UV, and they also suffer membrane damage by TiO2-NOAA itself, as a result, they reserve energy sources. From the result of Real Time RT-PCR, genes related to oxidative stress (GRE2, SOD2) were up-regulated in Condition 1 and 3, however, these expression levels in each condition were not significant. And genes related to synthesis of trehalose and glycogen (GSY1, TPS2) were up-regulated in Condition 1 and 3. These results suggest that oxidative stress is caused not by TiO2-NOAA but by UV. It is also suggested that yeast cells were damaged at their membranes by TiO2-NOAA, as a result, genes related to synthesis of trehalose and glycogen were up-regulated. Thus, we suggest that the effect of TiO2-NOAA on yeast cells under UV irradiation is greater due to TiO2-NOAA itself than due to ROS generated by TiO2-NOAA.当下，纳米颗粒已广泛应用于各类商业产品中。其中最为常见的纳米颗粒之一便是二氧化钛（titanium dioxide, TiO₂），其具备催化活性与紫外吸收能力（波长λ<400 nm），且可产生活性氧（reactive oxygen species, ROS）。二氧化钛纳米颗粒的催化活性可杀灭多种微生物。在自然环境中，纳米颗粒会形成由原生颗粒、凝聚体与附聚体构成的结构，此类聚集体被定义为NOAA（nano-objects, and their aggregates and agglomerates greater than 100 nm）。二氧化钛纳米颗粒的独特性质可在环境中得以保留，因此其应用规模的不断扩大引发了人们对其环境风险的担忧，评估TiO₂-NOAA的生物与生态效应具有重要意义。在我们此前的研究中，我们以酿酒酵母（Saccharomyces cerevisiae）与大肠杆菌（Escherichia coli）为实验对象，评估了TiO₂-NOAA对微生物的影响。实验结果表明，TiO₂-NOAA可在紫外辐照下分解亚甲基蓝，这表明TiO₂-NOAA在紫外辐照下会产生活性氧。但在紫外辐照条件下的基础琼脂培养基中，TiO₂-NOAA并未表现出生长抑制效果；当在培养基中添加TiO₂-NOAA后，即便使用可灭活微生物的紫外辐照强度，仍可观察到菌落形成。此外，TiO₂-NOAA可吸附微生物。上述结果表明，TiO₂-NOAA产生的活性氧含量不足以灭活微生物，且TiO₂-NOAA可能对微生物起到了紫外防护作用。本研究中，我们以酿酒酵母为实验对象，对TiO₂-NOAA的效应进行了更为细致的评估。我们采用DNA微阵列分析（DNA microarray analysis）开展定性评估，同时针对DNA微阵列分析中筛选出的特征基因，采用实时荧光定量逆转录聚合酶链反应（Real Time RT-PCR）法进行定量评估。为对比不同条件下的酵母细胞，我们设置了6组处理条件：1. 紫外辐照下与TiO₂-NOAA结合的吸附组分；2. 紫外辐照下未与TiO₂-NOAA结合的非吸附组分；3. 无紫外辐照时与TiO₂-NOAA结合的吸附组分；4. 无紫外辐照时未与TiO₂-NOAA结合的非吸附组分；5. 单独紫外辐照组；6. 阴性对照组。DNA微阵列分析结果显示，第1组的基因表达变化数量最多，其次为第3组与第5组。第1组与第5组的酵母细胞中，与氧化应激相关的基因表达显著上调；第1组与第3组的酵母细胞中，与海藻糖及糖原合成相关的基因表达显著上调。上述结果表明，紫外辐照下的TiO₂-NOAA会使酵母细胞遭受氧化应激，而TiO₂-NOAA本身则会对酵母细胞膜造成损伤，进而促使酵母细胞储备能量物质。实时荧光定量RT-PCR结果显示，第1组与第3组中与氧化应激相关的基因（GRE2、SOD2）表达上调，但各组的表达水平均未达到显著差异；而第1组与第3组中与海藻糖及糖原合成相关的基因（GSY1、TPS2）表达上调。上述结果表明，氧化应激并非由TiO₂-NOAA引发，而是由紫外辐照导致。同时，研究结果也表明，TiO₂-NOAA会对酵母细胞膜造成损伤，进而促使海藻糖与糖原合成相关基因的表达上调。综上，我们认为，在紫外辐照条件下，TiO₂-NOAA本身对酵母细胞的影响要强于其自身产生的活性氧所带来的影响。