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From 2013.igem.org

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Revision as of 10:07, 15 August 2013

The neurotoxic effects of acrylamide have been widely known for many years. Local symptoms of acrylamide poisoning such as mucous membranes and skin irritation have been formerly reported. There exists also systemic effect characterized by fatigue, sleepiness and memory difficulties; in extreme cases also hallucination, disorientation and confusion.

While acrylamide was perceived as severe neurotoxin, there had long not been enough studies on it’s possible carcinogenicity. Yet, the resent studies have showed that acrylamide may also be engaged in that kind of processes. Due to the rapid development of plastic industry, acrylamide is becoming to be even more widely present in our environment. But the manufacturing is not the only way, that acrylamide appears in our proximity. Because of Maillard reaction taking place while deep-frying or baking between an amino acid and a reducing sugar, acrylamide comes into being.

Possible ways of absorption of acrylamide are as follows: inhalation, ingestion, and through the skin and mucous membranes. Basing on a report of WHO from 2002 average daily intake for the general population ranges from 0.3 to 0.8 microgram of acrylamide per kilogram of body weight.

Increased consumption of deep-fried food and, following on from that, increased intake of acrylamide, may have serious health consequences. Recent studies have revealed that there exists an association between consumption of deep-fried foods and increased prostate cancer risk.1 What is also suggested, is that there is a weak positive correlation between increased acrylamide intake and increased possibility of occurrence of endometrial and ovarian cancer.2

Considering the alarming effects of the acrylamide exposure, we have decided to enclose to our project cytotoxity and genotoxity study. Our aim is to show how acrylamide may affect different tissues reflected by the cell lines derived from that tissues. In our experiments we would work on Hela and HEK cell lines representing ovarian and kidney cancer cells respectively.

To conduct our experiments we would implement following methods: Flow cytometry, Hoechst dyeing, Alamar Blue assay, and Micronuclei test.

Using flow cytometry we would be enabled to conduct precise analysis of chemical and physical parameters simultaneously. The measurement bases on cell sorting and cell counting according to chosen parameters. (…)

In order to be able to examine cell’s nuclei morphology we introduced staining with Hoechst dye. The dye binds to the minor groove of dsDNA preferentially to sequences rich in adenine and thymine. As the Hoechst dye may be excited by ultraviolet light and then emits blue fluorescent light we’ll be able to observe changes in the cell’s nuclei using fluorescent microscopy. As UV light is harmful for the cells, they have to be fixed first in order to avoid excess death during the assay. Our aim is to study changes that occurred due to treating cells with the acrylamide: cell division, cell apoptosis and amount of micronuclei.

To indicate cell viability, we have decided to implement Alamar Blue assay. It bases on the ability of living cells to convert nonfluorescent resazurin to the highly fluorescent molecule- resorufin. While entering the cells, resazurin is being reduced to resorufin and the bright, red fluorescence is to be observed. Viable cells are continuing to convert resazurin to resorufin, so the strength of fluorescence increases proportionally and may be used to measure viability of the cells.

As we would like to measure the genotoxity of acrylamide, we’ve decided to implement micronucleus assay. This test belongs to the most popular and widely used methods of searching for potentially mutagenic substances. The assay bases on formation of the micronuclei, which are the erratic nuclei formed during the anaphase due to mutagen exposition. They may originate both from acentric chromosome fragments or whole chromosomes unable to migrate properly. The modifications in chromosomes structure, observed during the test, indicate the genotoxic effect of tested substance.

In our study we would search for optimal acrylamide concentration for particular cell line in order to conduct the experiments. We expect to observing the cytotoxic effects in higher acrylamide concentrations i.e. 3-5 mM. In lower concentrations 1-2 mM may occur the elimination of the toxicant, which is to be examined.

Bibliography:

1. Stott-Miller M, Neuhouser ML, Stanford JL; Consumption of deep-fried foods and risk of prostate cancer; Epub 2013 Jan 17.

2. Ehlers A, Lenze D, Broll H, Zagon J, Hummel M, Lampen A; Dose dependent molecular effects of acrylamide and glycidamide in human cancer cell lines and human primary hepatocytes; Epub 2012 Dec 31

3. Ahmad Besaratinia_ and Gerd P.Pfeifer; A review of mechanisms of acrylamide carcinogenicity; Carcinogenesis vol.28 no.3 pp.519–528, 2007

4. O'Brien J, Wilson I, Orton T, Pognan F; Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity; Eur J Biochem. 2000 Sep;267(17):5421-6.

5. Naoki Koyamaa,b,c, Hiroko Sakamoto a, Mayumi Sakuraba a, Tomoko Koizumi a, Yoshio Takashima a, Makoto Hayashi a, Hiroshi Matsufuji b, Kazuo Yamagata b, Shuichi Masudac, Naohide Kinae c, Masamitsu Honmaa,; Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells; Mutation Research 603 (2006) 151–158