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, HEK 293 and 143b cell lines representing cervical cancer, human embryonic kidney and Bone osteosarcoma TK deficient cells respectively.

In our experiments we are implementing the following methods: Flow Cytometry, AlamarBlue assay, Cell Growth Assay and Caspase assay.

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

As a point of departure for our experiments we have decided to implement AlamarBlue 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. AlamarBlue assay gives reliable and repeatable results yet is uncomplicated to conduct. It serves as a trustworthy basis for further research.

In order to obtain repeatable and reliable results of experiments we have decided to conduct preliminary assays to determine plating density and concentration of acrylamide for each cell line: HeLa, HEK293 and 143b. The protocol of the assay as well as detailed conduct is to be found in the section: Extras.

The outcome of AlamarBlue assay showed that there exists strong, negative correlation between acrylamide concentration, time of incubation and cell viability. So the longer exposure time and the higher concentration of acrylamide, the weaker the cell viability is.

These results made us to consider further investigation. As we wanted to state whether loss of cell viability is caused by cell death or rather by limitation of cell growth and proliferation we decided to implement Caspase-Glo 3/7 Assay, Cell Growth Assay and Flow Cytometry.

Moreover, as the results of AlamarBlue assay for lower acrylamide concentrations (i.e. 1mM) seemed unclear, we decided to conduct additional assays for concentration ranging from 0,2 to 1mM. Those results are to be seen in section: Notebook

Results

Conduct of AlamarBlue assay showed that there is a strong, negative correlation between time, acrylamide concentration and cell viability for each of the cell lines: HeLa, HEK293 and 143b.

For 1mM concentration, distinct changes are to be observed after 48 (for HEK293) or 72 hours (for HeLa and 143b). For higher acrylamide concentrations, loss of cell viability is to be observed right away after 24hours with over 80 percent of dead cells after 96 hours.

The graphs presented below serve as the summary for each cell line:

Carrying out AlamarBlue assay for concentrations of acrylamide between 0,2 and 1mM showed that additional cell activity exist leading to removal of toxicant. Yet those results need to be confirmed and extended research on that issue need to be implemented.

In order to be able to state whether the loss of cell viability, that we have observed, was caused by decrease in cell division (so the cytostatic effect of acrylamide) or by cell death (so the cytotoxic effect of acrylamide) we have implemented Cell growth assays. To conduct the assays we have seeded cells from different cell lines (HeLa, HEK293 and 143b) in 6-wells plates adding proper concentration of acrylamide to each well and counted the amount of cells after 24, 48 and 72 hours. The results show that a limitation in cell growth occur after adding the acrylamide, and that amount of dead cells is connected to the acrylamide concentration.

The graphs presented below serve as the summary for each cell line:

Measurement of caspase activity with Caspase-Glo 3/7 Assay obtained from Promega, showed that there is a positive correlation between caspase activity and concentration of acrylamide. These result suggests that there is an increase in apoptosis for cells treated with higher acrylamide concentrations.

The graphs presented below serve as the summary for each cell line:

Results obtained from Flow Cytometry showed that the growth in amount of apoptotic and necrotic cells is caused by longer acrylamide exposure time and higher acrylamide concentration.

Summary

Despite the fact that it is impossible to relate directly the results obtained for the cell lines to human organism, it may give a basis for further examination and extended research. In our experiments we showed that there exists cytotoxic and cytostatic effect of acrylamide on HeLa, 143b and HEK293 cell lines. We also proved that there is a correlation between time of exposure to acrylamide and the decrease in cell viability and proliferation.

By carrying out our cytotoxicity study we wanted to emphasize the importance of synthetic biology research. After showing the possible harmful effects of acrylamide we wanted to reveal how products of synthetic biology may influence and facilitate our everyday life. We also wanted to raise the awareness of the threats connected with the consumption of tertiary processed foods.

Finally, let us make you imagine that you are able to check the amount of acrylamide before eating your chips. With our FluoSafe sensor it will be soon possible.