Materials And Methods Reports Examples

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Introduction

Nanoparticles are already in use in fairly wide domain of Science & Technology, as well as in medicine. Nanoparticles, as the name suggest are the minute particles in the nanometer range and their USP is their application in those areas where larger particles cannot be used. Due to their minuscule size, it is quite likely that they would be very highly reactive when used in conjunction with biological systems and probably affect the human health. It is true that ultrafine particles (may be bit larger than nanoparticles) have already been known to produce harmful inhalative effects. So in all likelihood, exposure of nanoparticles should produce the undesirable heath consequences.( Lison 2009).
A wide body of research, knowledge and awareness is present in the domain of environmental sciences, which confirm the adverse effects of ultrafine particles post inhalation. Obviously this happens when the proximity of ultrafine particles is too dangerously close to the human contact, as the ultrafine particles are integral parts of certain biological systems in use. As per the research nanoparticles also represent similar properties and reactivity as shown by the ultrafine particles. (Gonzalez, et. al., 2009)
Nanoparticles (NP) have distinct properties which are advantageous and disadvantageous due to several reasons. For one, they benefit many an aspect of our life, but at the same time it is definitely required that their risks are critically examined, as their toxicological effects may potentially affect the health. It is quite crucial to assess the potential negative effects of NP based products as these products have been available for general use and many more such products are likely to come in near future. Hence assessing their effects and toxicology with respect to the human health bear a prime importance. (Lison 2009).
There is an increased importance of studies and research which helps to understand as to how the NP based products can possible interact with the body of a human being due to their exposure either at work-place or at homes. Specific areas of interests are the NP cellular interactions at biological barriers. Incidentally, there is an FP7 project called NANOTEST which shares some details about the NP cellular interactions at biological barriers in context with the toxicological profile of NPs. (Gonzalez, et. al., 2009)
It has been informed in the several researches that the NPs cardio vascular effect has been seen in epidemiological studies. The research points out that these ultraparticles can easily permeate the cross body membranes and can travel from one organ to other through the membranes. Since nanoparticles are even smaller so evidently enhanced concentration of such particles from the respiratory epithelium, can move to circulation. This may cause their circulation to other vital parts of the body like heart, liver, spleen, brain and may even result in the cardiovascular dysfunction, blood coagulation, and can potentially affect the nervous system. (Thomassen, et. al, 2010)
Clearly the nanoparticles can permeate the organ membranes and travel to any part of the body causing serious health concern. As far as the commercial products are concerned where the nanoparticles are being used, major ones are like the sunscreens which use metal oxide nanoparticles. Sunscreens are used for UV protection and there are many similar products commercially available. (Gonzalez, et. al., 2009)

This lab experiment pertains to the analysis of frequency of chromosomal harm brought by presence of genotoxic material in mammal’s cell. There are following assumptions and properties associated with the genotoxis, which are primary to the context of this experimentation:
1. When genotoxin leads to the formation of adduct or lesion in the DNA, then the adduct would possibly be recuperated before replicating; however, if not repaired, then this can result in break in chromosome called Clastogenicity.
2. When a damage is caused in a component of the cells which are engaged in replicating process, then this can lead to the missegregation of the chromosome. The consequent daughter cell are termed as aneuploid; this means that the resulting cell may have more or less number of the chromosomes as required for this cell. (Thomassen, et. al, 2010)
3. When this condition of aneuploid occurs then mitosis should happen in order to detect the discrepancy in the chromosome. Mitosis results in generating the next generations of interphase-cell. This allows to detect the damage as micronucleus (Mn) is formed since the broken fragment or lost chromosome cannot attach to the spindle apparatus and will lag during the segregation of the chromosome. Consequently, Damaged DNAs will get enclosed to the own micronucleus (Mn).
4.Number of Mns formed or their frequency relates to the level of damage done, which itself depends on the level of exposure to NPs.
5. At this stage it is not viable to determine that whether the interphase cell has gone mitosis or not. So this is an inherent problem.
6. This is also one of the objects of the experimentation

The methods which are applied for this experimentation as follows:

1. To find the difference whether the cell has gone the division or not, the cell is treated with a test material and after this another chemical is administered which helps to prevent Cytokinesis. This compound is called Cy t o c h a l a s i n B.
2. by the administration of this compound, the binucleate cell (Bn) results. This helps us to understand that the cells which have undergone divisions would form Bn. Thus, in this way we can determine that which cells have undergone division.
3. The cells which have not gone the division will only form the mononuclear cells. This can be seen in the below figure:
4. The difference or the frequency of Mn and Bn gives us a fair idea on the level to which toxicities have occurred. Also how much dose of the chemical produces the given degree of damage, can also be determined with this experimentation.
5. Also the percentage of Bn gives a fair idea of the cytotoxicity of the test material as some substances may resist the growth of the cell or cause the death of the cell.

Aim of the Lab

In this lab, the object is to examine the human lymphoblastoid cell which has been administered various samples of NPs called super-magnetic iron oxide nanoparticles (U S P I O N). These samples are as follows:

Sample 1- - - negative control : 1O µm Poly styrene beads

Sample 2 - - - 50 µm diameter extra n coated USPION
Sample 3 - - - -600 µm diameter extra n coated USPION
Sample 4 - - - - Positive control : Methyl nitro sourea
Procedure
Coded slide is provided in which I scored 1000 cell approximately and classified them as mono / B i - / m u l t i - n u c l e a r. This slide helps to score the Bn cells and determine the percentage of such cells.
As it was highlighted this percentage of the Bn cells represent the proportion of those cells which have undergone divisions and thus represent the cytotoxicity of the samples as given above.
Also, it is to be determined that in 500 Bn cells, how many Mn cells are present; so this gives us the indication of the level to which the chromosomal harm has been inflicted with respect to the treatment.
The slides are then sorted out as per the order and the data is then added to the sheet and the chart is drawn.

Results

The slides represent the Bn and Mn cells and these slides were sorted out and the chart was made of the data. These charts are given below:

Data Summary Sheet

In vitro Micronucleus Assay
Data Summary Sheet
In vitro Micronucleus Assay
Data Summary Sheet
In vitro Micronucleus Assay
Discussion
The 4 set of graphs respectively for each charts are given below:
The x axis represents the data from 0-50
The statistical analysis is as follows:

References

Kirsch-Volders M, Gonzalez L, Carmichael P, Kirkland D. (2009). Risk assessment of genotoxic mutagens with thresholds: A brief introduction. Mutat Res. 678:72-5.
Gonzalez L, Thomassen LC, Plas G, Rabolli V, Napierska D, Decordier I, Roelants M, Hoet PH, Kirschhock CE, Martens JA, Lison D, Kirsch-Volders M. (2010). Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models. Nanotoxicology 4:382-95.
Gonzalez L, Sanderson BJS and Kirsch-Volders M. (2011). Adaptations of the in vitro MN assay for the genotoxicity assessment of nanomaterials. Mutagenesis, 26, 1, 185-191
Gonzalez L, Decordier I and Kirsch-Volders M. (2010) . Induction of chromosome malsegregation by nanomaterials. Biochemical Society transactions 38(6):1691-7
Napierska D, Thomassen LCJ, Lison D, Martens JA, Hoet PH. (2010) . The nanosilica hazard: another variable entity. Part Fibre Toxicol 7:39
Rabolli V, Thomassen LCJ, Princen C, Napierska D, Gonzalez L, Kirsch-Volders M, Hoet PHM, Huaux F, Kirschhock CEA, Martens JA, Lison D. (2010). Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles. 4:307-18.
Thomassen LCJ, Aerts A, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, (2010). Synthesis and Characterization of Stable Monodisperse Silica Nanoparticle Sols for in Vitro Cytotoxicity Testing. Langmuir 26:328-35.
Gonzalez L., Thomassen L, Plas G, Rabolli V, Napierska D, Decordier I, Hoet P, Kirschhock C, Martens J, Lison D and Kirsch-Volders M. (2009). Aneugenic and clastogenic effects of amorphous silica nanoparticles in A549 lung carcinoma cells: Size matters?. EMS meeting in St. Louis, Missouri, USA, 24-28.
Lison, D. (2009). Toxicology of Nanomaterials : Silica nanoparticles and Carbon nanotubes. Gaiker COST conference on the Toxicology of Nanomaterials, Bilbao.
Lison, D. (2009). Should we worry about the health risks of nanos ? IMEC Leuven.
Napierska, D. Thomassen L.C.J., Gonzalez L, Rabolli V, Lison D, Kirsch-Volders M, Martens JA, Nemery B, Hoet PH. (2010). Oxidative stress of amorphous monodisperse silica nanoparticles in humanendothelial cells. Nanosafe: International Conference on Safe Production and Use of Nanomaterials; Grenoble, France.
Thomassen LCJ, Napierska D, Rabolli V, Gonzales L, Kirch-Volders M, Hoet PH, Lison D, Kirschhock CEA, Martens JA. (2010).Nanotoxicology: the marriage of chemistry and biology. Systematic investigation of silica nanoparticle toxicity. At Dipartimento di chimica I.F.M., Turin, Italy.