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 cytotoxicity study. Our aim is to show how acrylamide may affect various cell lines originating from different tissues. In our experiments we are working on HeLa, HEK293 and 143B cell lines representing cervical cancer, human embryonic kidney and Bone osteosarcoma cells respectively.

In our experiments we are implementing the following methods: Flow Cytometry, AlamarBlue assay, Cell Growth Assay, Cell Cycle 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, 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 state how acrylamide may influence the cell cycle we've decided to implement Cell Cycle Assay. This method employs flow cytometry so as to envision phases of the cell cycle. Permeabilization of cells with cold ethanol and addition of propidium iodide (PI) prior to analysis, allows us to measure the fluorescence. PI binds to DNA quantitatively so the intensity of the fluorescence of the stained cells is correlated with the amount of DNA that they contain.

We expect to observe the cytotoxic effects in higher acrylamide concentrations i.e. 3-5 mM. In lower concentrations <1 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

Results

After conducting our experiment we have obtained following results:

• loss of metabolic activity (results obtained from AlamarBlue assay)

• increase in apoptotic cell death (stated by the presence of AnnexinV positive cells and the observation of caspase activity)

• suppression of cell growth and proliferation (stated during Cell Growth assay)

• disturbances in the cell cycle (results basing on the outcomes of Cell Cycle assay)

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.

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.

Flow Cytometry allowed us to reliably confirm obtained outcomes. Implementation of this method enabled us to observe changes in amount of apoptotic and necrotic cells for different acrylamide concentrations and incubation time, thus proving the cytotoxic effect of the acrylamide.

Figures for the Flow Cytometry

HeLa cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

143B cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 2mM, 5mM

HEK293 cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

In our study we've included Cisplatin control, whose results are to be seen in section: Notebook

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.

By carrying out Cell Cycle assay we were enabled to bring under scrutiny changes in the cell cycle. The conclusion that is to be drawn, is that there occur disturbances in the cell cycle depending on concentration of acrylamide and incubation time.

Histograms for Cell Cycle assay

HeLa cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

143B cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 2mM, 5mM

HEK293 cell line upper row: 24h incubation bottom row: 48h incubation acrylamide concentrations from left: NC, 1mM, 3mM, 5mM

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.

All of the results as well as graphs, figures and statistics are to be seen in section: Cellular biology lab journal

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.