Neurotoxic acrylamide

Acrylamide (AA) is formed during the cooking of plantbased foods when the free amino acid asparagine reacts with reducing sugars such as glucose and fructose in the Maillard reaction.
Acrylamide forms primarily in plant-based foods including potato products such as French fries and potato chips; cereal-grain-based foods such as cookies, crackers, breakfast cereals, and toasted bread; and coffee.
Acrylamide formation usually occurs at elevated temperatures when frying or baking (above 120°C (248°F)) and in low moisture conditions. However, acrylamide formation has been identified in some fruit and vegetable products heated at lower temperatures or higher moisture conditions (Amrein et al. 2007; FDA 2006a; FDA 2006b; Roach et al. 2003).
Acrylamide levels can vary widely between similar products, due to small changes in ingredients and in processing conditions.

AA is a colorless and odorless crystalline solid. Owing to its capability to polymerize it is commonly used as flocculant for water purification, a constituent in cosmetics and as a sealing adjuvant in tunnel construction.
Food Administration and the University of Stockholm reported considerably high levels of this probably carcinogenic compound in commonly consumed foods such as bread, coffee, potato crisps, French fries and many others.

AA is accidentally discovered in starchy foods like crisps, chips, and bread. Its discovery in heated-foods received great attention of public and medical community.

The International Agency for Research on Cancer in 1994 classified acrylamide as “probably carcinogenic to humans” (group 2A) (International Agency for Research on Cancer, 1994).
The European Food Safety Authority (EFSA) confirmed in 2015, that acrylamide is a carcinogen (EFSA CONTAM Panel, 2015).
Due to the toxic effect ofacrylamide on human health, European Commission has issued a recommendation to monitor, in all Member States, the acrylamide content in selected groups of the food products. The latest European Commission regulation on the monitoring of acrylamide in food has been published on November 20, 2017 and a reference levels of acrylamide was set at 400 μg/kg for roasted coffee whilst for instant coffee this was 850 μg/kg Commission Regulation (EU) 2017/2158, 2017). According to the above, many analytical methods have been developed to determine of acrylamide in food among others in coffee, which is one of the most popular beverages in the world.

AA shows several toxic properties, including genotoxicity, carcinogenicity and

AA exerts genotoxic effects in cells through oxidative DNA damage induced by the build-up of intracellular reactive oxygen species (ROS) and the depletion of glutathione (GSH) (Jiang et al., 2007). I

Additional research has indicated that AA causes neurotoxicity by inducing oxidative stress and activating cytochrome P450 2E1 (Ghanayem et al., 2005; Huang et al., 2012;
Lakshmi et al., 2012); the subsequent mitochondrial dysfunction triggered by these processes contributes to the neurotoxicity (Prasad and Muralidhara, 2013, 2014).

AA, a food contaminant, belongs to a large class of structurally similar toxic chemicals, ‘type-2 alkenes’, to which humans are widely exposed. Besides, occupational exposure to acrylamide has received wide attention through the last decades. It is classified as a neurotoxin and there are three important hypothesis considering acrylamide neurotoxicity: inhibition of kinesin-based fast axonal transport, alteration of neurotransmitter levels, and direct inhibition of neurotransmission. While many researchers believe that exposure of humans to relatively low levels of acrylamide in the diet will not result in clinical neuropathy, some neurotoxicologists are concerned about the potential for its cumulative neurotoxicity. It has been shown in several studies that the same neurotoxic effects can be observed at low and high doses of acrylamide, with the low doses simply requiring longer exposures.

General population can be mainly exposed to AA through consumption of carbohydrate-rich food processed at high temperatures (above 120 ◦ C). AA is formed spontaneously during heat-induced non-enzymatic reaction, also known as the Maillard browning reaction, between reducing sugars (glucose and fructose), and free amino acids (mainly asparagine) (Tareke et al., 2002). Groceries such as: coffee, chocolate, almonds, French fries, crackers, potato chips, cereal and bread typically contain the highest levels of AA (Tareke et al., 2002). In addition to that, the presence of AA is detected in used frying oil and it is also a component of cigarette smoke (JECFA, 2005; Hays and Aylward, 2008). The average AA intake for the general population and high consumers is estimated to be approximately 1 and 4 mcg/kg per body weight a day (mcg/kg bw/day), respectively (JECFA, 2005). More importantly, it is anticipated that the children have 2–3 times higher AA intake than the adults (FAO/WHO, 2002; Dybing et al., 2005; JECFA, 2005). Even more, AA is contaminant of baby food and infant formulas (Erkekoğlu and Baydar, 2010). The later consequently emphasized a concern for the health of a younger human subpopulations regarding dietary AA exposure. The whole spectrum of changes and consequences caused by AA intake, especially in young, developing organism are still poorly understood.

Baking is a complex process inducing physical, chemical and biochemical changes in the cereal matrix such as volume expansion, evaporation of water and formation of a porous structure, denaturation of proteins, starch gelatinization, crust formation and development of a desirable taste and pleasant flavours and browning. One of the main chemical reactions involved in the generation of those colours and flavours is the Maillard reaction, essential via for the production of acrylamide.

AA induces tumors in several organs in mice and rats and exerts reproductive and neurotoxic damage. After dietary consumption AA is rapidly absorbed from the gastrointestinal tract and widely distributed to the tissues. In the liver AA is metabolized to glycidamide (GA), which is more reactive towards DNA and proteins.
At low doses, 50% of acrylamide is metabolized to a DNA reactive metabolite, named ‘glycidamide’ by cytochrome 2E1 (CYP2E1)-mediated epoxidation. However, this conversion is saturable and at high doses only 13% of acrylamide is biotransformed to glycidamide. Glycidamide can be metabolized by epoxide hydrolase and both acrylamide and glycidamide can undergo conjugation with glutathione (GSH). The results of different studies support the concept of acrylamide by itself not acting as a genotoxic agent at exposure levels to be expected from food ingestion. Metabolic formation of glycidamide is essential for genotoxic effectiveness. Thus, glycidamide acts like the ultimate genotoxic metabolite of acrylamide and it was found to form DNA adducts and induce micronuclei in different organs of mice.
Acrylamide is a well-recognized potent neurotoxin affecting both the central nervous system (CNS) and peripheral nervous system (PNS). The magnitude of the toxic effect depends on the duration of exposure and the total exposure dose.

AA is formed mainly from free asparagine and reducing sugars during high-temperature cooking and processing of common foods, principally through Maillard reactions.

It is evident that the main sources of human dietary exposure to AA are those of fried potato, bakery products, breakfast cereals and coffee.

AA is mainly formed during heat processing (>120 ◦C) of foods – primarily those derived from plant origin such as potato and cereal products.

In mammalian organisms, AA is considered to be predominantly conjugated to glutathione (GSH) by spontaneous and/or glutathione-S-transferase (GST) mediated coupling. Reduced GSH plays an important role in detoxification processes taking part mainly in the liver (Kedderis 1996).
In addition, it was determined that acrylamide reduced the levels of cerebral GSH and inhibited the action of cerebral glutathione S-transferase (GST).

Acrylamide was suggested to inhibit several proteins (including a myriad of enzymes and motor proteins) and we may postulate that this may further contribute to axonal degeneration or dysfunction.

The dangers of high levels of acrylamide have been recognised for about a decade, but in 2015 the European Food Safety Authority published a risk assessment saying that high levels of acrylamide in food potentially increased the risk of developing all types of cancer in all age groups.

Due to reports of neurotoxic, genotoxic and carcinogenic activity of acrylamide, the presence of this compound in food may pose a risk to human health (Bergmark, 1997; Bull, Robinson, Laurie, Stoner, Greisger & Stober, 1984; Chapin, Fail, George, Grizzle, Heindel, Harry et al., 1995; Friedman, Dulak & Stedman, 1995; Johnson, Gorzinski, Bodner, Campbell, Wolf, Friedman et al., 1986; Lehning, Persaud, Dyer, Jortner &
LoPachin, 1998; LoPachin, 2004; Tyl, Marr, Myers, Ross & Friedmann, 2000).

AA has neurotoxic effects and is also carcinogenic in experimental animals. Human studies have revealed that occupational exposure to AA produces symptoms of peripheral neuropathy. Studies using hemoglobin adducts as biomarkers of occupational exposure of Swedish tunnel workers to a grouting agent have shown that AA resulted in mild and reversible peripheral nervous system symptoms. Likewise, a previous study in China involving 71 workers in a small AA factory showed that longer exposure to AA resulted in cerebellar dysfunction, followed by neuropathy.

AA is classified by the International Agency for Research on Cancer as probably carcinogenic to humans.

After dietary consumption, AA is rapidly absorbed from the gastrointestinal tract and widely distributed to the tissue. In the liver it is metabolized to an epoxide glycidamide by the liver metabolizing system CYP2E1. The metabolite glycidamide is far more reactive toward DNA and proteins than AA. Studies on rats, mice and humans suggest efficient human metabolism of AA to glycidamide. However, in humans there is considerable variability in the extent of AA conversion to glycidamide, which appears to be related to inter-individual variability in the amount of liver CYP2E1. Regarding the haemoglobin adduct levels of AA and glycidamide in humans compared to animal species Vikstrom et al. found that the AA haemoglobin adducts is about five times higher and glycidamide haemoglobin adducts nearly two times higher after dietary AA exposure compared to the levels in F344 rats exposed as in cancer studies; for glycidamide haemoglobin adducts the difference in biotransformation between humans and rats is regarded as modest. Fennell et al. concluded that internal exposure to glycidamide in humans was two to four times less than in the mouse. When applying water with 0.5, 1 and 3 mg kg−1 of AA to five volunteers 34% of the amount was recovered as total metabolites in the urine within 24 h. Fuhr et al. gave volunteers a meal containing 0.94 mg AA and recovered 60% of the administered dose as urinary metabolites within 72 h. Glycidamide may be further metabolized by epoxide hydrolase to glyceramide or by conjugation to glutathione, or it may react with proteins, including haemoglobin, or with deoxyribonucleic acid (DNA).

The current investigation in biscuits indicates that the use of wholegrain increases nearly 50% the average acrylamide content compared with refined grains, although no significant differences were observed. Similarly, biscuits with higher levels of protein and fibre presented higher content of acrylamide, but without significant differences.

Due to the higher prevalence for the celiac disease over the world, gluten-free products commercialisation has grown at an annual rate of 28%.30 These products are
formulated with gluten-free cereals and other ingredients, offering alternative foods intended for people with this disease. In this study, biscuits with and without gluten exhibited similar acrylamide content and also those classified as dietetic biscuit, where the low fat and/or sugar content did not significantly affect to acrylamide levels.

As expected due to the acrylamide levels found in the different groups of biscuits, rye-based biscuits represent the highest exposition level, followed by teff, spelt and oat based ones, ranging from 30.60 to 8.38 µg/day. Consumption of biscuits made of cereal mixture as well as those mainly formulated with rice and wheat leads to a median acrylamide exposure (4.18 – 3.23 µg/day), while a low exposition scenario takes place if buckwheat or corn-based biscuits are selected (2.44 -1.91 µg/day).

Mechanism of formation
The Maillard reaction, in the presence of asparagine, has been shown to be the main pathway for AA formation in a wide range of foods processed at high temperatures.
AA is mainly formed during heat processing (>120 ◦ C) of foods – primarily those derived from plant origin such as potato and cereal products. Stable isotope-labeled experiments have shown that the backbone of the AA molecule originates from the amino acid asparagine. Asparagine alone could in principle form AA by direct decarboxylation and deamination, but the reaction is inefficient, with extremely low yields.

Most important for AA formation in the Maillard reaction is the presence of its precursors in raw materials, e.g. reducing sugar such as glucose and fructose, and amino acids in the form of free asparagine, and the magnitude of the combined temperature and time load. Although Maillard reaction is the main mechanism of AA formation, depending on the type of food raw material the reaction may present differences in terms of the limiting reactant. In this sense, some genetic factors as well as environmental conditions may also affect the level of AA precursors. Additionally, the processing conditions and water activity of foods may also influence AA formation.

In general, the content of asparagine is higher in whole-grain flour than in the sifted fractions. Therefore whole-grain products may contain a higher content of AA.

AA is also formed during roasting of coffee beans, a Maillard reaction process where at the same time the color and the aroma of the coffee beans are produced. The precursors of AA formation are asparagine, which is the amino acid with the second highest concentration in green coffee beans, and carbohydrates, of which sucrose is present in the highest amount. Sucrose is found in concentrations up to 9% dry weight in green coffee beans and during roasting it splits into the reducing sugars glucose and fructose. Asparagine is the limiting precursor for AA formation and is found in higher amounts in Robusta coffee compared to Arabica coffee, implicating higher AA amounts in Robusta compared to Arabica coffee.

Decaffeinated samples were found to contain up to 3-fold more AA compared with caffeinated samples of the same brand. This variation may be attributed to the decaffeination process that entails five steps (Chu et al., 2013). In the decaffeination process, the swelling of beans is carried out in water after which caffeine is extracted. Stream stripping is applied to remove all solvent residues from the beans. Finally, the adsorbents are regenerated and the decaffeinated coffee beans dried to their initial moisture content (Chu et al., 2013). In short, the decaffeination process subjects the coffee beans to higher temperatures for longer periods of time. As a result, the acrylamide content in decaffeinated products is enhanced, rendering the decaffeinated alternative product a high source of the carcinogenic and neurotoxic AA.

Coffee is a main contributor to the human dietary intake of acrylamide, a chemical known to exhibit carcinogenic and neurotoxic risks (Guenther et al., 2007). In March 2018, a California judge ruled that “coffee firms must add cancer warning on their products”. This verdict was based on the presence of toxic chemicals in coffee, of which only acrylamide was mentioned (CBSLA, 2018; Deabler, 2018; Turner, 2018).

In addition to the precursors, AA formation in foods will depend on the heating conditions (heat, time) and water activity of the products. With increasing temperature the reaction rate of AA formation increases in most cases. However, the formation of AA in coffee peaks some time before the coffee is ready roasted and the concentration in the roasted coffee is somewhat lower than at the peak level. AA boils at 193 ◦ C and typical roasting temperature is 210–250 ◦ C. Hence some elimination by evaporation or degradation takes place during the roasting and, for that reason, dark-roasted coffee contains less AA than medium-roasted coffee.

It is clear that acrylamide is neurotoxic in animals and humans. The neurotoxic effects, however, seem to be only a problem in humans with high-level exposure.

Imprtantly, AA could not be detected in unheated or boiled foods.

Author Artūras

1. Pedreschi, F., Mariotti, M. S., & Granby, K. (2013). Current issues in dietary acrylamide: formation, mitigation and risk assessment. Journal of the Science of Food and Agriculture, 94(1), 9–20. doi:10.1002/jsfa.6349. 
2. Kovac R, et al. Acrylamide alters glycogen content and enzyme activities in the liver of juvenile rat. Acta Histochemica (2015),
3. Kumar J, Das S and Teoh SL (2018) Dietary Acrylamide and the Risks of Developing Cancer: Facts to Ponder. Front. Nutr. 5:14. doi: 10.3389/fnut.2018.00014.
4. BMJ 2017;356:j354 doi: 10.1136/bmj.j354 (Published 2017 January 23).
5. Erkekoglu, P., & Baydar, T. (2013). Acrylamide neurotoxicity. Nutritional Neuroscience, 17(2), 49–57. doi:10.1179/1476830513y.0000000065. 
6. Wawrzyniak, R., & Jasiewicz, B. (2019). Straightforward and rapid determination of acrylamide in coffee beans by means of HS-SPME/GC-MS. Food Chemistry, 125264. doi:10.1016/j.foodchem.2019.125264. 
7. Zhao, M., Dong, L., Zhu, C., Hu, X., Zhao, L., Chen, F., & Chan, H. M.(2019). Proteomic profiling of primary astrocytes and co-cultured astrocytes/microglia exposed to acrylamide. NeuroToxicology. doi:10.1016/j.neuro.2019.09.005. 
8. M. Mesías, F. J. Morales and C. Delgado-Andrade, Food Funct., 2019, DOI: 10.1039/C9FO01554J.
9. El-Zakhem Naous, G., Merhi, A., Abboud, M. I., Mroueh, M., & Taleb, R. I. (2018). Carcinogenic and neurotoxic risks of acrylamide consumed through caffeinated beverages among the lebanese population. Chemosphere, 208, 352–357.
10. Abt, E., Robin, L. P., McGrath, S., Srinivasan, J., DiNovi, M., Adachi, Y., & Chirtel, S. (2019). Acrylamide levels and dietary exposure from foods in the United States, an update based on 2011-2015 data. Food Additives & Contaminants: Part A, 1–16.

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