Potential Toxicological Effects And Mechanism Of Zinc Oxide Nanoparticles On Silkworm (Bombyx Mori)

Nanotechnology is a new technology that provides a great possibility of novel application in the fields of biotechnology and agricultural products.With the rapid development of today's nanoscience technology,more and more nano materials are used widely in the industry field and life,which greatly increased the chance of the nano materials contact with humans,therefore,the safety of nano materials has attracted wide attention,the Zinc Oxide nanoparticles(ZnO NPs)as one of the most widely used nano materials used in sunscreen and skin care industry,its biological safety is always the focus of people's attention.The study on the toxicity mechanism of Zinc Oxide nanoparticles mostly based on cells,bacteria,mice and other model organisms,and the toxicity mechanism is not very clear,especially the toxicity of whole organisms.Less research of nano toxicology mechanism based on the whole mice,therefore,with the short growing time,less experimental animal safety,mammals ethics and other advantages compared to mice,the silkworm as a new model organism were used to study the acute toxicity of nano Zinc Oxide research.This experiment adopt the way of acute abdominal injection of Zinc Oxide nanoparticles suspension into silkworm,the lethal dose(LD50)and the toxicity of Zinc Oxide nanoparticles to Bombyx mori was determined,appropriate concentration around semi lethal dose was used to evaluate the acute toxicity to silkworm.The transmission electron microscopy(TEM)and scanning electron microscopy(SEM)were used to characterize the Zinc Oxide nanoparticles.The growth status of silkworm after 48 h exposure were observed,body weight and food digestion were determined.The silkworm midgut,silk gland,fat body and trachea were observed and analyzed by histopathology.The inductively coupled plasma mass spectrometry(ICP-MS)was used to determine the content of zinc in the midgut,silk gland,fat body and trachea respectively in 60 h after exposure,compared the nano Zinc Oxide accumulation transport of various tissues and organs,determine the potential target organs of nano Zinc Oxide in silkworm.The midgut total protein content,superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)were determined to evaluate the oxidative damage in the midgut.qRT-PCR was used to analyze the apoptosis relating genes Dronc,Caspase-1 and Trt of silkworm.The results showed that the semi lethal dose of Zinc Oxide nanoparticles of the 5instar larvae of each silkworm was 737.31 ?g,according to the classification standard of GHS,the toxicity was the third grade.When the silkworm was infected with 12 h,toxic symptoms such as convulsions,cuticle shrinkage and fasting appeared,the body weight of the experimental silkworm larvae was significantly lower than that of the control group since 12 h investigation.Then pathological observation of the midgut,silk gland,fat body and trachea cells in 48 h showed different degrees of damage and rupture.The ICP-MS results showed that the concentration of zinc in the midgut was the highest,the concentration of the group was 5.8,10,28.1 and 33.2 times respectively of the trachea,fat body,blood and silk gland.The concentration of zinc in the midgut increased significantly from the 12 h,and reached the maximum at 48 h as time went on.For the rest organs,the zinc element results showed a downward trend as a whole,so the midgut of the silkworm was identified as the potential target organ of the Zinc Oxide nanoparticles.So the midgut was selected as the experimental object to study the toxicological mechanism.The activity of the SOD enzyme relating to the production and clearance of reactive oxygen species showed the rise trend in the beginning 36 h,this may help the body resist a large number of oxygen free radicals.then decreased in the later 12 h.After the silkworm was poisoned,CAT and GSH enzymes activity showed the opposite trend in 36 h,at 36-48 h,the two enzymes activity decreased,the trend was the same as that of SOD.It is speculated that the production of reactive oxygen species leads to the silkworm cell apoptosis,thus the relative quantitative analysis of apoptosis relating genes Dronc and Caspase-1 in silkworm were used,the two genes were the upstream promoter and effect genes of Caspase apoptosis pathway in silkworm,the relative expression of Dronc and Caspase-1 showed the rise trend with the exposure time went by,The experiment group of the gene Bmtrt showed a downward trend,which indicated that the telomerase was inhibited after the silkworm midgut cells infection,which may related to the silkworm apoptosis after the silkworm infection.This study provides a reference for studying the acute toxicity mechanism of nanomaterials using silkworm as a special model animal.