人造奈米碳黑微粒於非細胞系統及動物毒性研究

Abstract

隨著奈米科技的發展，人造奈米材料被廣泛的應用在科學、技術及醫學上。奈米材料可以使用在一些較大粒徑微粒所無法達到的特殊目的上，但奈米材料跟較大粒徑微粒相比較，可能具有更高的生物活性。奈米微粒可以懸浮在空氣中經由呼吸、食入或直接穿透皮膚進入人體，也有可能進入地下水系統改變生物活性、進入食物鏈，對生物及環境造成衝擊，目前針對奈米材料的環境、安全及衛生規定尚未完備。 最近的研究顯示人造奈米微粒造成健康危害可能與反應性氧化物種（reactive oxygen species, ROS）有關。因此本研究將探討奈米微粒ROS的產生與微粒粒徑大小及表面積之相關性，並進一步以自發性高血壓大鼠（spontaneous hypertensive rat, SHR）及健康的SD（Sprague Dawley）大鼠動物模式，探討奈米微粒的暴露與氧化壓力、肺部發炎反應之相關。 非細胞系統利用15、51及95nm的奈米碳黑微粒（ultrafine carbon black, ufCB），於200、400及800μg/ml下暴露一小時，再以2',7'-dichlorofluorescin（DCF）螢光分析法評估ROS的產生。動物實驗部分，分別以SHR及SD大鼠為實驗動物，以氣管灌注15、51及95nm的ufCB 500及1000μg，並以磷酸生理食鹽水（phosphate buffered saline, PBS）為控制組，暴露24小時後犧牲，取肺泡灌洗液分析肺部發炎指標；另外分析血清8-hydroxy-2'-deoxyguanosine（8-OHdG）及周邊白血球DNA單股斷裂（DNA single-strand breaks, DNA SSB）情形。 實驗結果顯示，在非細胞系統中，各種粒徑之ufCB ROS的產生，隨重量濃度增加而增加，在相同重量濃度下，粒徑越小的CB產生的ROS越高，有趣的是，總表面積與ROS產生有高度相關（R2＝0.83）。動物實驗部分，氣管灌注1000&micro;g ufCB的SHR，與控制組比較，暴露15nm肺泡灌洗液中嗜中性球百分比顯著增加（P<0.05），暴露51及95nm則沒有顯著差異；暴露15nm之SHR，其血清8-OHdG有增加的趨勢，雖然無統計上顯著差異，此外，周邊白血球DNA SSB tail monent及％ of tail length亦較控制組高（P<0.05），然而暴露51及95nm的氧化傷害情形反而隨著暴露劑量增加而降低。以SD大鼠為實驗動物部分，研究結果發現暴露15nm之SD大鼠，其肺泡灌洗液中總細胞數及嗜中性球百分比都隨著暴露劑量增加而增加，且暴露高、低劑量組與控制組比較，嗜中性球百分比都有顯著較高（P<0.05）；但是在氧化傷害部分，8-OHdG及DNA SSB都沒有顯著升高的情形。進一步綜合比較氧化壓力傷害指標及各發炎指標的相關性，在SHR部分發現實驗動物肺泡灌洗液中總細胞數與血清8-OHdG有顯著相關（R2=0.23，P<0.05），而肺泡灌洗液中嗜中性球百分比與周邊白血球DNA SSB各參數指標也都有達到統計上顯著相關（tail moment R2=0.36、％ of tail length R2=0.27、% of tail intensity R2=0.25，P<0.05）；另外，血清8-OHdG與周邊白血球DNA SSB各參數指標也有顯著相關（tail moment：R2=0.6、％ of tail length：R2=0.44、% of tail intensity：R2=0.43，P<0.05）。在SD大鼠部分，血清8-OHdG與肺泡灌洗液中的乳酸脫氫酵素（lactate dehydrogenase, LDH）有顯著相關（R2=0.21，P<0.05），血清8-OHdG、DNA SSB與其它發炎指標則沒有顯著相關。比較SHR與SD大鼠肺部發炎情形及血清8-OHdG的差異，不論是實驗動物的基本值、隨暴露劑量或總表面積增加的趨勢，SHR肺泡灌洗液中總細胞數及血清8-OHdG都比SD大鼠來得高；以控制組校正後，肺泡灌洗液中總細胞數隨暴露劑量增加的趨勢，SHR也較SD大鼠來得高，各DNA損傷指標隨暴露劑量增加的趨勢，在暴露最小粒徑微粒之SHR也都較SD大鼠來得高。 總結來說，本研究發現奈米微粒能產生ROS，而ROS的產生與微粒總表面積有關，動物實驗部分證明奈米微粒會增加體內ROS的產生及肺部發炎反應，在SHR易感性動物實驗發現暴露ufCB會加重肺部發炎及氧化傷害的表現，且與微粒總表面積有關。本研究也指出易感性動物對於肺部發炎及氧化傷害的危險性增加，這部份具有重要的政策意涵，但需要有更多進一步的研究去釐清其相關機制。

Nanoparticles are increasingly used in science, technology and medicine, and they are produced for specific purposes which cannot be met by large particles and bulk material. However, recent evidences reveal that nanoparticles are likely to be highly reactive with biological systems. Currently environmental, healthy and safety regulation regarding to nanoparticles have not be well-established. Recent studies indicated that artificial nanoparticals-induced health effects may be associated with the generation of reactive oxygen species (ROS). However, the exact relationship remains unclear. In order to investigate the possible mechanism, we used a cell-free system study to verify the relationship between particle size, total surface area and ROS generation. Further, we investigated the effects of nanoparticle exposure on oxidative stress and pulmonary inflammation on spontaneously hypertensive rats (SHR) and Sprague Dawley (SD) rats. In cell-free system, ultrafine carbon black (ufCB) with average diameter of 15, 51 and 95nm were suspended in phosphate buffered saline (PBS) in 200, 400 and 800 μg/ml for 1hr. DCF (2’,7’-dichlorofluorescin) assay was used to determine the ROS generation of ufCB. In animal study, SHR and SD rats were intratracheally instillation of 15, 51 and 95nm ufCB in 500 and 1000μg, separately. Animals administrated PBS were treated as control group. Animals were sacrificed 24hr after treatment. Bronchoalveolar lavage fluid (BALF) was collected for pulmonary inflammation analysis. Serum 8-OHdG (8-hydroxy-2'-deoxyguanosine) and peripheral blood DNA single-strand breaks (DNA SSB) were determined to evaluate the effects of oxidative stress. Our results revealed that the generation of ROS increased with the instilled mass concentration in each particle size. At the same mass concentration, smaller particle size of CB produced greater ROS. Interestingly, the generation of ROS was highly correlated with total surface area of particles (R2=0.83). In animal study, SHR treated with 1000&micro;g of 15nm ufCB had significantly increased the proportion of neutrophils in BALF as compared to SHR with larger particle treatment. No significant increased pulmonary inflammation was observed in SHR treated with 51 and 95 nm ufCB at same dose. SHR with 15nm ufCB treatment demonstrated increased serum 8-OHdG, although it did not reach statistical difference. In addition, SHR treated with 15nm ufCB showed significant increased DNA SSB in tail moment and ％ of tail length as compare to the controls (P<0.05). However, the DNA SSB was reduced with the increasing ufCB treatment with 51 and 95 nm in SHR. In SD rat model, we found the proportion of neutrophils in BALF was significantly increased in SD rats treated with 15nm ufCB as compared to the controls (P<0.05). However, there was no significant elevation of serum 8-OHdG and peripheral blood DNA SSB after ufCB treatment. In SHR, interestingly, we found correlation between oxidative stress and pulmonary inflammation, BALF total cells correlated with serum 8-OHdG (R2=0.23; P<0.05) and the proportion of neutrophils in BALF correlated with peripheral blood DNA SSB (tail moment R2=0.36, % of tail length R2=0.27, % of tail intensity R2=0.25; P<0.05). We also found that serum 8-OHdG statistically correlated with peripheral blood DNA SSB (tail moment R2=0.6, % of tail length R2=0.44, % of tail intensity R2=0.43; P<0.05). In SD rats, serum 8-OHdG correlated with BALF LDH (lactate dehydrogenase) (R2=0.21; P<0.05). However, there was no significant association found between 8-OHdG, peripheral blood DNA SSB and other pulmonary inflammation indicators. Serum 8-OHdG and BALF total cells at baseline or their trends for either exposure dose or total surface area in SHR was higher than those in SD rats. After adjusting for control, the trend of BALF total cells in SHR was higher than those in SD rats. However, the trend of DNA damage indicators, which was adjusted for control, in SHR was higher than those in SD rats only for the exposure to 15nm nanoparticles. In summary, we found nanoparticles generated ROS in cell free system and the generation of ROS was associated with total surface area of particle. In vivo study found that exposure to nanoparticles caused ROS generation and pulmonary inflammation. In SHR susceptible animal study we found that exposure to ufCB would increase burden on lung inflammation and oxidative damage which were related to total surface area of particles. Our study also indicates that susceptible animals maybe subject to increased risk of lung inflammation and oxidative DNA damage. This may have important policy implication. However, more studies are needed to clarify the above findings.