Overview

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

While acrylamide was perceived as a severe neurotoxin, there had long not been enough studies on its possible carcinogenicity. Yet, the recent research have shown that acrylamide may also be engaged in this kind of processes. Due to the rapid development of the plastic industry, acrylamide is becoming even more widely present in our environment. But the manufacturing area is not the only way by which acrylamide appears in our proximity. While deep-frying or baking, through a chemical reaction between an amino acid and a reducing sugar (called the Maillard reaction), 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 micrograms of acrylamide per kilogram of body mass.

Increased consumption of deep-fried food and, subsequently, 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. 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.

Considering the alarming effects of acrylamide exposure, we have decided to include in our project a cytotoxity and genotoxity study. Our aim is to show how acrylamide may affect different tissues reflected by the cell lines derived from these tissues. In our experiments we are working on HeLa and HEK 293 cell lines representing cervical cancer and human embryonic kidney cells respectively.

In our experiments we are implementing the following methods: flow cytometry, Hoechst dyeing, Alamar Blue assay, and Micronuclei test.

To indicate cell viability, we have decided to implement the 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.

Flow cytometry enables us to conduct precise analysis of chemical and physical parameters simultaneously. The measurements bases on cell sorting and cell counting according to chosen parameters. After conducting flow cytometry experiments we will be able to state whether treated cells are undergoing apoptosis, necrosis or if the observed metabolic effects are only cytostatic.

In order to be able to examine cell nuclei morphology we introduced staining with the 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 will be able to observe changes in the cell’s nuclei using fluorescent microscopy. As UV light is harmful to 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 number of micronuclei.

As we would like to measure the genotoxity of acrylamide, we have 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 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 are searching for apropriate acrylamide concentration for particular cell line in order to be able to observe the cytotoxity. We expect to observe 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, whose effects are 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

Getting the experiments underway