摘要
由于具有良好的光电、催化、超导性能和杀菌消毒活性,纳米银已经成为目前市场上应用最广泛的金属纳米材料,商品化产品几乎无处不在。纳米银的大量生产和广泛应用增加了其向环境释放的机会,同时增加人体暴露的机会。因此,纳米银颗粒对生物及生态系统的影响日益得到关注。本课题采用水相合成法制备粒径为25-30 nm的纳米银颗粒,利用小型鱼medaka动物模型分别评价纳米银颗粒的急性、亚慢性毒性和胚胎发育毒性以及组织分布特征,并探讨了纳米银颗粒毒性作用机制。由于自由态银离子是目前公认的最强鱼类毒性金属之一,而纳米银在环境或生物体内可以释放Ag+,本实验还通过对照实验比较了两种不同形态银的组织分布和毒性效应。
采用次磷酸盐液相还原法制备纳米银干粉,合成体系中加入聚乙烯吡咯烷酮(PVP)作为稳定剂,通过有机溶剂洗涤、离心以及真空干燥等过程获得纯净纳米银干粉。XRD衍生光谱表明制备产物为金属银,符合面对心(face-centered cubic, fcc)立方体银晶体结构。紫外-可见分光光度计(UV-vis)测定纳米银颗粒的最大吸收波长为400 nm,不仅表明球形纳米银颗粒,而且显示纳米银颗粒在制备和分散过程中没有发生团聚和氧化。透射电镜和粒度分析表明纳米银粒径分布均匀,绝大多数颗粒大小为25-30 nm。总的来说,本实验中获得分散相良好、粒径分布均匀、稳定的纳米银颗粒有利于生物效应及毒性作用评价。
在急性毒性实验中,纳米银颗粒在24 h内的UV-vis光谱特征无明显变化,而且不改变溶解氧含量,成年medaka静水暴露96 h的LC50值为0.87 mg/L。在亚慢性毒性实验中,成年medaka连续静水暴露14 d,较低浓度组(0.05-0.25 mg/L)受试动物死亡率与对照组无统计学差异(P>0.05),最高暴露组(0.5 mg/L)medaka死亡率达37.5%,与对照组有明显差异(X2=7.05,P<0.01),纳米银对medaka的亚慢性最大无致死剂量为0.1 mg/L。纳米银主要造成肝脏、鳃和肠道金属负荷的增加,尤以肝脏中银含量增加最明显。生化分析显示纳米银诱导肝细胞损害,造成肝、鳃等组织中超氧化物歧化酶(SOD)、过氧化物酶(CAT)活性下降以及还原型谷胱甘肽(GSH)浓度减少,同时造成脂质过氧化产物含量明显增加,表现氧化损伤效应。组织病理学检查也证实纳米银暴露造成肝脏和鳃组织损伤。综合纳米银在成年medaka体内的组织分布和产生的毒理学终点,肝脏是纳米银颗粒的毒作用靶器官。此外,相对于急性毒性实验和死亡率指标,medaka亚慢性毒性实验的组织病理损伤对纳米银毒性比较敏感,可以作为灵敏的生物标志物来评价金属纳米颗粒生态毒性。
众所周知,生命早期小型鱼对化学物质暴露和环境应激比较敏感,本部分实验采用目前通行的部分生命周期实验(Partial-life test)检测纳米银的胚胎毒性。在70 d暴露期间,较高浓度纳米银颗粒(400-1000μg/L)明显抑制medaka胚胎的发育过程,比较典型的毒性包括发育延迟、色素细胞减少、视顶盖最大宽度(脑发育指标)减少以及血液缓流等。胚胎期的观察还发现纳米银暴露造成高发生率的形态发育异常和畸形,包括水肿、骨骼畸形、心脏异常、眼睛发育异常以及鳍膜缺损等等。实验中得到的发育迟滞和畸形发生率的非线性剂量-反应关系反映了纳米银颗粒毒性作用的复杂性。在实验中我们还发现大量水肿病及淤血的medaka胚胎孵化后常常伴随形态发育异常,因此推测纳米银暴露造成胚胎期medaka渗透压调节紊乱,而淤血意味着血循环系统输送营养素质能力的下降,这些可能是造成胚胎期medaka发育异常的主要作用机制。本实验提示体内实验是评价纳米银颗粒毒性作用的敏感实验方案,同时部分生命周期实验可以有效地评价纳米银颗粒的发育毒性和生态危害。
依据胚胎发育毒性实验,我们通过光学显微镜、扫描电镜技术、组织生化分析以及自由基测定探讨了纳米银胚胎毒性的作用机制。纳米银颗粒可以破坏medaka卵壳表面结构和完整性,损害卵壳的机械保护作用,同时降低卵壳维持卵内外环境渗透压平衡的能力,这些可以从机理上解释水肿病高发和胚胎发育毒性。纳米银暴露造成卵内乳酸脱氢酶(LDH)活性明显增加,表明medaka胚胎处于缺氧环境,而缺氧会影响需氧生物的能量代谢及正常发育。根据卵内SOD活性、还原型GSH含量、脂质过氧化产物-丙二醛(MDA)浓度、活性氧族(ROS)以及单线态氧的变化,纳米银还造成medaka胚胎产生氧化应激。这些生物效应或毒性效应主要发生在胚胎发育早期,随着胚胎期幼鱼的发育,纳米银颗粒所致的部分毒性效应具有恢复趋势。虽然如此,由于生命早期淡水鱼(如胚胎、孵化后幼鱼)对化学物质暴露和环境应激比较敏感,此时期幼鱼发生严重和不可逆损伤肯定影响水生鱼类群落。
纳米银表面容易被氧化,在水、体液、生物体内很容易释放离子态银(Ag+),而后者影响着纳米银的生物效应和毒性作用。采用银离子选择电极法测定不同含量纳米银溶液中Ag+的含量,数据表明新鲜制备的溶液中银阳离子比例为0.94%-1.01%,释放率与时间成非线性增加。利用成年medaka亚慢性毒性实验比较银离子含量相当的纳米银与Ag+的组织分布、死亡率、氧化应激效应和组织病理损伤特征,结果显示两种形态金属银的组织分布特点和毒性效应各不相同。在Ag+含量相同的情况下,纳米银的毒性较弱。
Silver nanoparticles (AgNPs) have taken on wonderful and unique chemo-physical virtues and biological properties that differ from those of their macro scaled or ionic counterparts. These differences have led to ubiquitous applications of silver nanoparticles (AgNPs) in optoelectronics, catalysis system, medical fields and personal healthcare. Presently, nano-silver becomes the most common group of metal engineered nanomaterials in commercial products. The increasing uses of AgNPs in consumer products have increased their release to the environment and, meanwhile, resulted in their exposure to living tissues. This would thus promote special concerns of environmental risks and human health associated with these particles.
Some evaluations have been carried out to demonstrate the plausible deleterious effects of nanoscale silver on microorganisms, algae, freshwater fish and rodent animals, however, the information of the AgNPs toxicity and environmental risks remains unclear. Silver nanoparticles that show considerably changed physical, chemical and biological properties increase these complex influences caused by exposure to AgNPs in the environment. In addition, the impossibility of release of ionic silver (Ag+) also should be taken into account to understand the AgNPs toxicity. Present work was performed to decipher in vivo biological activity of Ag nanoparticles on adult medaka fish and embryonic larvae as well as to explain the mechanisms for their toxicity. With a comparative study, we also described the discrepancy between nanoscale silver and ionic silver.
The colloidal silver nanoparticles were prepared by reducing a high molar concentration of silver nitrate solution (up to 1 M) with sodium hypophosphite in the presense of polyvinylpyrrolidone 30 (PVP 30). Purified powder of AgNPs was subsequently produced through solvent cleaning, centrifugation and drying in vacuum oven to remove the residual ingredients. We have utilized X-ray diffraction (XRD), UV-vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) spectrometry to characterize the silver nanoparticles obstained. These data showed spherical shape and face-centered cubic (fcc) phase of AgNPs with shallow size distribution ranged from 20-35 nm, and no agglomeration and oxidation of silver nanoparticles were observed. The experimental AgNPs exhibited a homogeneous dispersion in aqueous solutions. The stable state of these silver nanoparticles should ensure that biological or toxicological activity in medaka model may specially attribute to nanoparticles rather other components and chemical mixtures.
In the acute toxicity tests, we found that the UV-vis spectrum pattern of AgNPs showed unchanged and the dissolved oxygen (DO) displayed consistency during 24 h AgNPs exposure. The caculated 96-h LC50 was at 0.87 mg/L, based on the mortality of adult medaka exposed to different concentration of nanoscale silver. In sublethal toxicity tests, adult medaka was treated with 0-0.5 mg/L AgNPs for consecutive 14 days to investigate organ distribution. Meanwhile, biochemical assessment and histopathological analysis were performed to evaluate possible harmful effects in terms of antioxidant enzymes, lipid peroxidation and histopathology. Highest concentration of AgNPs significantly increased mortality of medaka to 37.5% during the exposure (X2=7.05, P＜0.01), though no statistic difference was found between low dose groups and control group. It's showed that AgNPs exposure enhanced the metal burden in gill, intestine and, specially, in liver. Dose-dependent decreases of Lactate dehydrogenase (LDH), Catalase (CAT) and superoxide dismutase (SOD) acitivities and total reduced GSH content were observed in liver, gill or brain of medaka exposed to Ag nanoparticles, however, increase of malondialdehyde (MDA) was induced in the AgNPs treated medaka-, suggesting that oxidative damage was induced by nano-Ag. Histopathological assay also revealed severe damages in the gill and liver. The results indicated liver of medaka could be the target organ of AgNPs toxicity.
Using Japanese medaka (Oryzias latipes) at early-life stages as experimental models, the developmental toxicity of silver nanoparticles was investigated following exposure to 100-1000μg/L homogeneously dispersed AgNPs for 70 days, and developmental endpoints were evaluated by microscopy during embryonic, larval and juvenile stages of development in medaka. Meanwhile, histopathological changes in the larval eye were evaluated. Retarded development and reduced pigmentation were observed in the treated embryos by AgNPs at high concentrations (≥400 ug/L). Maximum width of the optic tectum, as an indicator of midbrain development, decreased significantly in a dose-related manner. Furthermore, silver nanoparticles exposure at all concentrations induced a variety of morphological malformations such as edema, spinal abnormalities, finfold abnormalities, heart malformations and eye defects. Histopathological observations also confirmed the occurrence of abnormal eye development induced by AgNPs. The data showed non-linear or U-shaped dose-response patterns for growth retardation at 5 days of postfertilization, as well as the incidence of abnormalities. Preliminary results suggested that the developmental process of medaka may be affected by exposure to silver nanoparticles. Morphological abnormalities in early-life stages of medaka showed the potential developmental toxicities of silver nanoparticles. Further research should be focused on the mechanisms of developmental toxicity in fish exposed to silver nanoparticles.
Using light microscopy and scanning electron microscopy (SEM), the results indicated that AgNPs treatments at higher concentration (≥125μg/L) may severely destroy the surface ornamentation and egg envelope. Meanwhile, significant increase in lactate dehydrogenase (LDH) activity, an indicator of anaerobic metabolism, indicated hypoxia in treated groups. Biochemical changes in SOD activity, reduced GSH level, TRARS concentration showed oxidative stress caused by silver nanoparticles in early-stage medaka embryos. Unexpectedly, a weak dose-dependent reduction in levels of ROS and singlet oxygen by high AgNPs exposure (≥250μg/L) as observed. We also noted that responses by antioxidant defenses in well-developed embryos were elevated, but the developmental damages caused by silver nanoparticles showed no recovery. Overall, it suggested that the disturbed egg chorion, hypoxia and oxidative stress were mechanistically associated to AgNPs toxicity in embryonic fish.
Ionic silver may be easily released from silver nanoparticles whose surface oxidation occurred in water, medium, and specially, under biological conditions. Toxic effects of AgNPs may be related to interactions of Ag+ ions with proteins and enzymes. As measured by Ag-ISE (Ion-selective electrode), maximum free Ag concentrations present in the fresh AgNPs suspensions were 0.94%-1.01%, and the dissolved silver was time-dependent increased with non-linear pattern for 24 h. We have examined the sub-chronic toxicity of silver nanoparticles and Ag+ to adult medaka by evaluating the distribution, mortality of tested fish, oxidative stress endpoints and histopathological changes. When compared as a function of the free ionic silver levels, Ag+(AgNP3) resulted in much higher toxicity such as mortality and oxidative damages than that of AgNPs.
引文
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