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Abstract This study was carried out at the Department of Genetics (Drosophila Lab) and the Molecular Genetic Laboratory, Faculty of Agriculture, Ain Shams University, Cairo, Egypt, the Nanotechnology Center, Regional Center for Food and Feed, Agricultural Research Center (ARC), Giza, Egypt, the Regional Center for Mycology and Biotechnology, Al-Azhar University and the Molecular Services Laboratory (H316), Research Center, October University for Modern Sciences and Arts (MSA) during the period from 2012 to 2017. Silver nanoparticles (AgNPs) are widely used in a growing number of applications due to their unique properties Due to the intensive commercial application of silver nanoparticles (AgNPs), risk assessment of this nanoparticle is of great importance. There is a growing production and application of silver nanoparticles (AgNPs) in various areas including catalysis, consumer products, food technology, textiles/fabrics, as well as medical products and devices. It was reported that about 25% of the >1300 nanomaterial-containing consumer products contain AgNPs. The rapid growth in the commercial use of AgNPs will inevitably increase silver exposure in the environment and the general population. However, with the accelerating introduction of AgNPs into commercial products, there is likelihood of their release into the environment, which gives rise to health and environmental concerns. The aims of the present study were: 1.To investigates the impact of AgNPs exposure in D. melanogaster with regard to changes in the expression of heat shock (hsp) genes (hsp23, hsp26, hsp27, and hsp60). 2. To assess the correlations between hsps genes, antioxidant systems and oxidative stress as reflected by changes in the expression of antioxidant enzymes (SOD, CAT and GSH). 3. To assess the ability to tumors development by monitoring the changes in the expression of tumor suppressor gene (p53). 4. To detect some genetic biomarkers associated with biological stress in fruit flies. The results could be summarized as the following: (A) Physiochemical level 1- By using transmission electron microscopy (TEM): most of the silver nanoparticles AgNPs were shown to be in a spherical shape and with an average diameter of 15-70 nm. 2- By using dynamic light scattering (DLS): silver nanoparticles were found to be mono dispersed with hydrodynamic diameter 74.35 nm. (B) Toxicity results; 1- Larvae treated with six concentrations of silver nanoparticles (25, 100, 200, 400, 800 and 1600μg/ml), revealed a reduced larval and pupal survival, affected larval pigmentation, adult loss of melanin, defects in adults and the appearance of some malformations compared with the control. 2- Silver nanoparticles AgNPs stimulated the mortality rate of - Drosophila larvae. The average survival rate of the control (water) was 98%.In concentrations of (25 μg/ml), (100 μg/ml), (200 μg/ml),(400 μg/ml),(800 μg/ml) and (1600 μg/ml) 4%,6% ,10%, 16% ,36%, and 50% of larvae were killed, respectively. This allowed the determination of the value of “LC50” at a concentration of 1600μg / ml. 3- Different concentrations of silver nanoparticles during the larval stage resulted in cuticle defects in adults. At AgNPs of 800 mg/L and above, adult flies were appeared lighter in body color with little or no melanin pigments left in their body. AgNPs selectively interferes with the melanin pigmentation. 4- Increasing the concentration of Silver Nano-particles from 25 to1600 μg/ml inhibited cuticle sclerotization as indicated by the light color of the larvae.5- Larvae exposure to 800 μg/ml AgNPs and above, effectively caused melanization and pale abdomen of female in adult flies as compared to wild type flies 6- Treatment with AgNPs revealed dramatic phenotypic modifications in the subsequent generations of Drosophila resulted in production of abnormal adults inducing leg and wing abnormalities as well as deformations of wings, eyes, and thorax. (C) Biochemical level 1- DPPH assay; This assay was used to measure the activity of antioxidant molecules and resulted in; a) Decrease of antioxidant activity of larvae treated by silver nanoparticles. b) Increase of antioxidant activity of larvae treated by heat shock compared to larvae treated with silver nanoparticles. c) Decrease of antioxidant activity of larvae treated with silver nanoparticles, followed by heat shock treatment compared to treatment by heat shock only. d) DPPH free radical scavenging assay revealed that AgNPs reduce the antioxidant activity compared with control that may affect on the immune system due to low antioxidant activity. 2- Reactive oxygen species (ROS) assay; a) Larvae treated with silver nanoparticles showed an increase amount of ROS. b) Larvae treated with heat shock showed an increase of ROS compared to the control larvae. c) Larvae treated with silver nanoparticles followed by heat shock treatment revealed a significant increase in ROS compared to the control larvae. (D) Molecular genetic analysis: Experiments were carried out with the use of RT-PCR using 2X SYBR® Green PCR master Mix assay to measure the relative changes in mRNA expression levels using four hsps genes (hsp23, hsp26, hsp27, and hsp60), three antioxidant genes (SOD,CAT and GSH) and tumor-suppressor gene (p53) in order to: 1- Assess the correlations between hsps genes, antioxidant genes and oxidative stress. 2- Assess antioxidant enzymes (SOD, CAT and GSH) 3- Assess tumor-suppressor gene (p53). a) The results revealed significant changes in the expression of the four hsp genes (hsp23, hsp26, hsp27, and hsp60) occurred in larvae after exposure to 1600μg/ml AgNPs followed by heat shock treatment. b) Gene expression was determined as the relative fold change normalized with RP49 mRNA expression. The expression of hsp26, hsp23, and hsp27 genes transcription level in the heat shock treatment were increased respectively (44.67-Fold), (15.21-Fold), (5.56-Fold), while the expression of hsp60 gene transcription level was decreased (0.29-Fold).The expression of hsp23, hsp60, and hsp27 genes transcription level in the AgNPs nano-stress treatment were decreased by (0.76-Fold), (0.53- Fold), (0.37-Fold), respectively. The expression of hsp26 gene transcription level was increased (4.65-Fold).While the expression of hsp26, hsp27, hsp23 and hsp60 genes transcription level in the AgNPs nano-stress+ heat shock treatment were increased by (13.34-Fold), (1.32-Fold), (1.19-Fold),(1.06-Fold), respectively. c) Antioxidants and tumor-suppressor gene (p53). The study revealed a significant increase in the gene expression of antioxidant genes (CAT, SOD, GSH) and tumor-suppressor gene (p53) as follows: a) The levels of gene expression due to exposure to heat shock were increased by (2.60-Fold), (2.14-Fold), (1.43- Fold), (1.14-Fold), respectively.b) The expression of p53 and GSH genes transcription levels in the AgNPs nano-stress treatment were increased by (16.03-Fold), (1.34-Fold), respectively. c) The levels of gene expression of SOD and CAT genes decreased by (0.59-fold) and (0.42-fold), respectively. d) The expression ofp53, GSH, CAT and SOD genes in the AgNPs nano-stress heat shock treatment were increased by (50.36-Fold), (1.84-Fold), (1.32-Fold) and (1.29-Fold), respectively. Finally, these results sound alarming; potential health and environmental effects of nanoparticles need to be thoroughly assessed before their widespread commercialization. Though there are few studies on cytotoxicity of nanoparticles on mammalian and human cell lines, there are hardly any reports on genotoxic and cytotoxic behavior of nanoparticles in plant cells. They underline the importance of systematic and reliable toxicology characterizations of nanomaterials and the necessity of extensive efforts by the nano-science community in designing and testing suitable nanoscale surface engineering/coating to develop biocompatible nanomaterials with no hazardous effects for human health and environment. |