الفهرس 
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Abstract
Nanotechnology is considered as one of the best key technologies for the future. This is mainly attributed to the fact that nanoparticles (NPs) exhibit completely different characteristics and functions in relation to the corresponding bulky materials with the same chemical composition. They are commonly used in products at the range of 1 to 100 nm. In this range, NPs achieve unique physicochemical properties which are widely accepted to be useful for the manipulation of a wide range of valuable end products for both civilians and armed forces.
Among other engineered nanomaterials, Ag-NPs have occupied a prominent position in the scientific as well as industrial community and being incorporated in a large number of military applications such as protective suits, combat tents, medical dressings, wound bandages, surgical meshes, and many others. Most of these products are in direct contact with the skin which represents a probable exposure route upon the release of these NPs from these products.
It is noteworthy that the same characteristic properties of NPs which have given them the advantage over traditional chemicals, may also led to exceptional hazardous effects, by creating the opportunity for their uptake and interaction with biological systems. In fact, although the toxicity of silver and its compounds is well documented, there are still gaps in the risk assessment of this precious metal in the nano form. Hence, the present study was intended to address the potential toxicity and the biological effects of Ag-NPs at both the physiological and molecular levels, following the dermal exposure to NPs.
During the current work, 45 male rabbits (Oryctolagus cuniculus) were used as an animal model. The design was arranged as acute (4 days) and sub-acute (7 and 14 days) exposure terms. Each experimental time period included 15 rabbits which were further divided into 3 levels (n=5): the control, exposure and recovery, respectively. In general, the suspension of test material was applied topically (1ml) as a single dose of 2.0% Ag-NPs for acute exposure and of 0.5% Ag-NPs every other day in the sub-acute term.
Several parameters were investigated including the quantification of intercellular accumulation and retention of Ag+ ions in the skin and their distribution in the other vital tissues such as plasma, liver, kidneys and spleen by using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) technique.
The effects of Ag+ free ions on the concentration of total proteins and the lipid profile were also evaluated in these tissues through specific spectrophotometric assays. In addition, the potential actions of Ag-NPs were evaluated in the skin by measuring the levels of different oxidative stress biomarkers such as GST, GR, GPx, GSH, SOD, CAT, MDA and H2O2. The 8-OHdG concentration was quantified an indicator for DNA oxidation.
Furthermore, the quantification as well as the gene expression profile of MMP-1, MMP-2 and MMP-9 were investigated in the skin. This was done in order to determine the effect of Ag-NPs on these proteolytic enzymes which, have been traditionally associated with the degradation and turnover of most components of ECM. These enzymes are essentially involved in many physiological processes such as inflammation and wound healing which are the main task of several Ag-NPs applications.
The physicochemical properties of Ag-NPs were fully characterized, where the particle size and shape were identified by the transmission (TEM) and scanning (SEM) electron microscopy, whereas the purity was examined through the EDX and XRF techniques. The examinations revealed the normal distribution of Ag-NPs within samples without any aggregates. The examined NPs were spherical in shape with an average particle size of 14nm and composed of 99.9% pure elemental silver.
The present data demonstrated the ability of Ag-NPs to overcome the skin barrier function and Ag+ ions were detected in all tissues under investigation. The spleen and liver seemed to be the main target organs for Ag-NPs with the highest accumulation levels followed by the skin, kidneys and the plasma at the both exposure terms. The acute term experimental results showed that the plasma Ag+ concentration rapidly declined in the recovery group followed by the skin, kidneys, liver and finally the spleen, but the elimination was not enough to bring the tissue concentrations back to the corresponding control levels.
This picture signified the ability to eliminate Ag+ from the different tissues in a relatively concise recovery period. Meanwhile, the sub-acute exposure data revealed the complete elimination of Ag+ ions from the skin and plasma after the recovery period following both exposure periods. The elimination of Ag+ ions, however, in the rest of tissues under investigation was less efficient.
The accumulated Ag+ ions did not exert significant effect on the concentration of total protein either in the spleen or kidneys of all experimental groups. In addition, no changes were observed in the protein level in the skin, plasma and liver during the sub-acute exposure for 7 days. In contrast, a highly significant decrease in total proteins was displayed in the skin and plasma of male rabbits at the exposure and recovery levels of both acute (2.0%, 4 days) and sub-acute (0.5%, 14 days) exposure to Ag-NPs, respectively. On the contrary, the liver exhibited an increased level of total protein during these exposure terms which may reflect its contribution to produce specific proteins for tissue repair or to support the cellular ability to combat the oxidative stress.
The current results also displayed the impact of Ag+ ions on the lipid profile. A significant increase in the total cholesterol, LDL-cholesterol and triglycerides were found following the acute exposure to 2.0% Ag-NPs concomitant with a reduction in HDL-cholesterol in all tissues under investigation, except the skins which was insignificantly affected. The spleen and liver exhibited the highest alterations in the lipid profile followed by the kidney and plasma, throughout the different levels of the acute exposure term. In general, the magnitude of perturbations in the tissues of the exposure groups was higher profound than that of the corresponding recovery groups.
The impact of Ag-NPs after the sub-acute exposure term seemed to be time dependent where, all the lipid parameters (Total, HDL-, LDL-cholesterols and triglycerides) were significantly affected in the tissues, after the 14 days exposure period. While, no significant changes were recorded in all examined tissues after the 7 days of exposure.
In the present work, the skin revealed strong signs of severe assault in response to oxidative stress. This was reflected by strong correlations between the examined antioxidant biomarkers and the accumulated Ag+ ions. The results demonstrated a remarkable elevation in the level of MDA and H2O2 concomitant with alterations in the GST, GR, and GPx activities in the skin after the dermal exposure to Ag-NPs in both acute and sub-acute experimental terms. In contrast, the skin showed a significant decrease in GSH and SOD contents along with a decline in CAT activity during these exposure terms. In general, the effects of Ag-NPs during the acute exposure term were significantly higher than those of the sub-acute exposure.
The disturbed antioxidant levels in the skin of exposed rabbits were significantly ameliorated during the recovery period, even though, they did not reach the control levels. These results altogether along with correlation and regression analyses pointed out to a prominent ability to overcome the stressor assault, eliminate Ag+ ions, and restore much of the biochemical homeostasis in a relatively short period of recovery.
The excessive generation of free radicals in the present work, led to a considerable oxidative DNA damage as indicated by the formation of 8-OHdG. There was a highly significant increase in the 8-OHdG concentration in the skin of the exposure and recovery groups throughout both acute and sub-acute exposure terms to Ag-NPs. In addition, the results of the correlation analysis confirmed the ability of Ag-NPs to aggravate oxidative DNA damage over time, which is considered as the main genotoxic pathway of these NPs. In the meantime, the positive correlation with the accumulated Ag+ ions might also suggest the possibility for restoring the regular levels of 8-OHdG and repair the DNA, if longer recovery time is allowed.
The molecular investigations also revealed the remarkable effect of Ag-NPs on the skin MMPs at both the concentrations and the transcriptional levels. The concentrations of MMP-1, MMP-2 and MMP-9 as well as the expression of their encoding genes were significantly elevated in the skin at the exposure and recovery levels of both acute and sub-acute experimental terms.MMP-9 was the most affected enzyme, whereas MMP-2 showed the least effect. Both the concentrations and gene expression of MMP-1 and MMP-2 were ameliorated to the control levels after the recovery period, whereas MMP-9 failed to return to the control levels.